**2. Protoporphyrin IX detection**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

hemoproteins (e.g. hemoglobin, myoglobin) [3, 4], are complexed to iron and occur ubiquitously. They are critical to life, because hemoproteins are involved in the transport of diatomic gases (respiration), chemical catalysis and electron transfer [5]. During heme synthesis from glycine and succinyl-CoA (**Figure 2**) a number of

*Porphyrin structures. Porphin (left), the simplest representative, and heme b (right), the prosthetic group of, e.g., hemoglobin, myoglobin, catalase, and cytochromeP450. Other members of the heme group (e.g. heme c in cytochrome C) differ slightly in the side chains. Porphyrins chelate divalent ions such as iron in heme. Due to their delocalized system of* π*-electrons they fluoresce after excitation. Different nitrogen forms in the pyrrole* 

Both ALA and PPIX have become of great interest to neurosurgery, because in gliomas, ALA diffuses into the tumor and induces PPIX-synthesis [6]. A surgical method has been developed taking advantage of both the enrichment of PPIX in the tumor

*Heme synthesis from succinyl-CoA to PPIX and ultimately heme. Synthesis of ALA is the rate-limiting step and under negative feedback control of heme (green/ dashed). A deficiency of iron supply limits heme synthesis and leads to PPIX accumulation. Zinc can then substitute for iron and ferrochelatase catalyzes the formation of zinc* 

intermediates including δ-aminolevulinic acid (ALA) are produced until, ultimately, protoporphyrin IX (PPIX) is converted to heme by insertion of a divalent

iron (Fe (II), catalyzed by ferrochelatase) [1].

*ring are labelled (red/italic, blue/bold).*

**56**

**Figure 2.**

*PPIX (red/dotted) [1].*

**Figure 1.**

#### **2.1 Erythrocyte protoporphyrin analysis**

Erythrocyte protoporphyrin (EP) served as a diagnostic marker for lead poisoning and environmental lead pollution as well as for iron deficiency anemia at the end of the 20th century [12]. From 1972 to 1991, it was officially recommended as the primary screening test for childhood lead poisoning by the Center for Disease Control and Prevention in the United States [13–15]. For the clinical diagnosis of porphyrias [16], rare disorders resulting from enzyme variability in heme biosynthesis, the porphyrin pattern is determined in blood, urine and faeces based on fluorescence techniques.

Taking advantage of the strong absorption of porphyrins in the Soret band (380– 430 nm) and their fluorescence, spectrophotometric and -fluorometric methods have been preferred for EP determination so far. The free erythrocyte porphyrin (FEP) test [17, 18], was, however, based on liquid-liquid extraction (LLE) at acidic pH, which dissociated zinc protoporphyrin (ZnPPIX) to metal-free PPIX during the extraction process. Thus, a sum parameter with different - and unknown - contributions of free PPIX and ZnPPIX was measured leading to false conclusions. The ratio of ZnPPIX to metal-free PPIX in erythrocytes varies, because in lead poisoning and iron deficiency anemia, ZnPPIX is accumulated in the blood, whereas in protoporphyria, the metal-free PPIX is elevated [12, 19, 20].

Hence, neutral ZnPPIX-specific LLE methods were developed, but they suffered from poor extraction efficiency [20, 21]. The widely applied ethyl acetate-acetic acid LLE method had three problems [17, 18]: First, the low extraction efficiency of PPIX from whole blood in comparison to the extraction from pre-diluted blood, which provided better precision of analysis; second, impurities in ethyl acetate influencing fluorescence and requiring pre-tests of reagent batches; third, the instability of PPIX standards prepared with deionised water. Thus, EP analysis required great attention to detail, because method modification, sample contamination or aging of standards and reagents had a great impact on analysis [22]. As a result, inter-laboratory comparison of EP results was generally poor while intra-laboratory precision was good [23].

#### **2.2 Spectrophotometry and –fluorometry**

In 1977, a hematofluorometer (HF) was designed for the detection of ZnPPIX in a drop of whole blood without sample pretreatment, which allowed immediate, simple and inexpensive detection [24]. Spectrophotometric and -fluorometric analysis became conventional analytical practice, but the inter-laboratory

agreement for EP levels was still poor and standardization has not been achieved [25]. PPIX levels measured with the FEP-test and HF-values of ZnPPIX did not match [13, 23]. Problems arose as a result of the limited PPIX stability towards light exposure, its tendency to form molecular aggregates in aqueous solution and buffer/ solvent-dependent variation in absorbance [13]. Consequently, different values have been published for the molar absorptivity of PPIX, leading to discrepancies in the calculation of PPIX and ZnPPIX concentrations from measured absorbance using Beer's Law or fluorescence emission intensity [13]. In addition, interferences such as bilirubin, increased hemoglobin, riboflavin, quinine as well as several drugs including doxorubicin or amoxycillin disturbed HF measurement; results improved with extended washing of erythrocytes [13, 26].

The application of dual-wavelength excitation in the HF technique [24] allowed the recording of background fluorescence, which then could be removed from the analyte spectra [27]. In 2019, it was shown that even the non-invasive measurement of erythrocyte ZnPPIX in children and women after childbirth was possible on the wet vermillion of the lower lip using this principle [28, 29].

The fluorescence method is rapid and easy to use, but it is limited by high background fluorescence. Complex algorithms for the processing of the obtained spectra are required. As fluorescence is measured as a sum parameter of all contributing analytes, results can easily be overinterpreted, especially when measuring at the lower limit of detection. This has to be taken into account when free PPIX is the target instead of ZnPPIX or the sum parameter of both. ZnPPIX is the predominant species of EP in circulating erythrocytes while free PPIX is only present in trace amounts [20, 30, 31]; the specific quantification of metal-free PPIX next to ZnPPIX is thus challenging.

#### **2.3 Liquid chromatography**

The absorption in the Soret band is broad and the resolution of absorption spectra is low resulting in spectral overlap. Chromatographic separation of porphyrins is thus recommended [25]. It isolates the analyte from the sample matrix and concentrates it. Conclusively, the application of high-performance liquid chromatography (HPLC) coupled with fluorescence detection (FLD, UV-visible detection was not sensitive enough) [30, 32–37] and MS became highly popular. Initially performed thin layer chromatography assays were gradually replaced in the 1980s [36].

HPLC-FLD was demonstrated in the differentiation of porphyrin species in porphyria [33, 37] or lead poisoning [36]. It is now the technique of choice for porphyrin analysis in routine and research laboratories [28, 36]. Methods are timeconsuming with typical elution times above 20 min [34, 35]. Moreover, the analysis of lipophilic PPIX remains challenging, because of its comparatively low recovery. Porphyrins respond quite differently in HPLC and attempts to measure them all in a single run have been abandoned. Porphyrins also have different excretion patterns in the body so that the majority of laboratories determines free PPIX and ZnPPIX in whole blood or plasma, and other porphyrins (uroporphyrin I and III, coproporphyrin I and III, **Figure 3**) in urine, using individual HPLC-FLD methods optimized for each purpose [28, 33–35, 37].

Advanced column media and higher pressure LC instrumentation (ultra highperformance (UHP) LC) further improved resolution and analysis time at lower solvent consumption although conventional HPLC was still widely applied. UHPLC from biological matrices like blood or tissue is not yet a routine technique in porphyrin analysis, because advanced sample preparation is required to avoid column contamination and clogging. Still, the use of UHPLC in conjunction with MS has great potential regarding sensitivity and speed of analysis [38].

**59**

**2.4 Mass spectrometry**

**Figure 3.**

tion [42] for the purpose.

extraction and single ion monitoring MS [42].

*positions 8 and 13 was not observed, for MS spectra see Figure 4).*

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

MS detection has a number of advantages over FLD [26, 27, 39]. Both high mass accuracy and the possibility of breaking the analyte molecules in the gas phase and measuring their fragments (MS/MS) contribute to unequivocal species identification, because complex samples can often not be fully chromatographically resolved. HPLC-MS was applied for the determination of porphyrin profiles in biological matrices [11, 39–43] using predominantly electrospray ionization (ESI) interfaces. Additionally, there were efforts to explore atmospheric pressure chemical ioniza-

*Structures of type I and III isomers of uro- and coproporphyrin in comparison to PPIX and mesoporphyrin* 

*(MPIX). Differences of type I and III isomers are highlighted in blue (uroporphyrin) and green (coproporphyrin). Differences of MPIX and PPIX structures at positions 8 and 13 are shown in grey. Fragmentation of PPIX in MS analysis is given in red (in the ion trap, cleavage of ethylic side chains at* 

In urine, uroporphyrin, coproporphyrin and other porphyrins were measured [41, 42]. As urine is a matrix with comparatively low complexity compared to blood and tissue, minimal sample pretreatment (acidification) was sufficient allowing high-throughput analysis. Providing excellent sensitivity and specificity by operating in multiple reaction monitoring mode, the presented method was superior to FLD [41]. Another approach in urine used porphyrin esterification followed by

Regarding porphyrin detection in more complex matrices, Sullivan and coworkers [39] quantified twelve porphyrins including PPIX in liver extracts based on their mass, because most porphyrins co-eluted. MS/MS was not carried out. The method suffered from problems with peak-splitting and matrix suppression [39]. Furthermore, highly acidic extraction was performed so that this approach was not

suitable for the individual determination of metal-free PPIX and ZnPPIX.

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

*Protoporphyrin IX Analysis from Blood and Serum in the Context of Neurosurgery… DOI: http://dx.doi.org/10.5772/intechopen.95042*

**Figure 3.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

with extended washing of erythrocytes [13, 26].

is thus challenging.

**2.3 Liquid chromatography**

optimized for each purpose [28, 33–35, 37].

great potential regarding sensitivity and speed of analysis [38].

wet vermillion of the lower lip using this principle [28, 29].

agreement for EP levels was still poor and standardization has not been achieved [25]. PPIX levels measured with the FEP-test and HF-values of ZnPPIX did not match [13, 23]. Problems arose as a result of the limited PPIX stability towards light exposure, its tendency to form molecular aggregates in aqueous solution and buffer/ solvent-dependent variation in absorbance [13]. Consequently, different values have been published for the molar absorptivity of PPIX, leading to discrepancies in the calculation of PPIX and ZnPPIX concentrations from measured absorbance using Beer's Law or fluorescence emission intensity [13]. In addition, interferences such as bilirubin, increased hemoglobin, riboflavin, quinine as well as several drugs including doxorubicin or amoxycillin disturbed HF measurement; results improved

The application of dual-wavelength excitation in the HF technique [24] allowed the recording of background fluorescence, which then could be removed from the analyte spectra [27]. In 2019, it was shown that even the non-invasive measurement of erythrocyte ZnPPIX in children and women after childbirth was possible on the

The absorption in the Soret band is broad and the resolution of absorption spectra is low resulting in spectral overlap. Chromatographic separation of porphyrins is thus recommended [25]. It isolates the analyte from the sample matrix and concentrates it. Conclusively, the application of high-performance liquid chromatography (HPLC) coupled with fluorescence detection (FLD, UV-visible detection was not sensitive enough) [30, 32–37] and MS became highly popular. Initially performed thin layer chromatography assays were gradually replaced in the 1980s [36]. HPLC-FLD was demonstrated in the differentiation of porphyrin species in porphyria [33, 37] or lead poisoning [36]. It is now the technique of choice for porphyrin analysis in routine and research laboratories [28, 36]. Methods are timeconsuming with typical elution times above 20 min [34, 35]. Moreover, the analysis of lipophilic PPIX remains challenging, because of its comparatively low recovery. Porphyrins respond quite differently in HPLC and attempts to measure them all in a single run have been abandoned. Porphyrins also have different excretion patterns in the body so that the majority of laboratories determines free PPIX and ZnPPIX in whole blood or plasma, and other porphyrins (uroporphyrin I and III, coproporphyrin I and III, **Figure 3**) in urine, using individual HPLC-FLD methods

Advanced column media and higher pressure LC instrumentation (ultra highperformance (UHP) LC) further improved resolution and analysis time at lower solvent consumption although conventional HPLC was still widely applied. UHPLC from biological matrices like blood or tissue is not yet a routine technique in porphyrin analysis, because advanced sample preparation is required to avoid column contamination and clogging. Still, the use of UHPLC in conjunction with MS has

The fluorescence method is rapid and easy to use, but it is limited by high background fluorescence. Complex algorithms for the processing of the obtained spectra are required. As fluorescence is measured as a sum parameter of all contributing analytes, results can easily be overinterpreted, especially when measuring at the lower limit of detection. This has to be taken into account when free PPIX is the target instead of ZnPPIX or the sum parameter of both. ZnPPIX is the predominant species of EP in circulating erythrocytes while free PPIX is only present in trace amounts [20, 30, 31]; the specific quantification of metal-free PPIX next to ZnPPIX

**58**

*Structures of type I and III isomers of uro- and coproporphyrin in comparison to PPIX and mesoporphyrin (MPIX). Differences of type I and III isomers are highlighted in blue (uroporphyrin) and green (coproporphyrin). Differences of MPIX and PPIX structures at positions 8 and 13 are shown in grey. Fragmentation of PPIX in MS analysis is given in red (in the ion trap, cleavage of ethylic side chains at positions 8 and 13 was not observed, for MS spectra see Figure 4).*

#### **2.4 Mass spectrometry**

MS detection has a number of advantages over FLD [26, 27, 39]. Both high mass accuracy and the possibility of breaking the analyte molecules in the gas phase and measuring their fragments (MS/MS) contribute to unequivocal species identification, because complex samples can often not be fully chromatographically resolved. HPLC-MS was applied for the determination of porphyrin profiles in biological matrices [11, 39–43] using predominantly electrospray ionization (ESI) interfaces. Additionally, there were efforts to explore atmospheric pressure chemical ionization [42] for the purpose.

In urine, uroporphyrin, coproporphyrin and other porphyrins were measured [41, 42]. As urine is a matrix with comparatively low complexity compared to blood and tissue, minimal sample pretreatment (acidification) was sufficient allowing high-throughput analysis. Providing excellent sensitivity and specificity by operating in multiple reaction monitoring mode, the presented method was superior to FLD [41]. Another approach in urine used porphyrin esterification followed by extraction and single ion monitoring MS [42].

Regarding porphyrin detection in more complex matrices, Sullivan and coworkers [39] quantified twelve porphyrins including PPIX in liver extracts based on their mass, because most porphyrins co-eluted. MS/MS was not carried out. The method suffered from problems with peak-splitting and matrix suppression [39]. Furthermore, highly acidic extraction was performed so that this approach was not suitable for the individual determination of metal-free PPIX and ZnPPIX.

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