**4.2 Gas phase fragmentation**

As already demonstrated in the literature for other porphyrins [40], PPIX ionizes as singly charged [M+H]+ species (*m*/z 563.3) in ESI-MS. MS/MS fragmentation 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 that is used as precursor for MS3 fragmentation which then generates further side 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 extended π-electron systems [50–52].

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 differ by four mass units in comparison to PPIX.

ZnPPIX was measured in MS/MS mode as it showed lower ionization efficiency than MPIX and PPIX. The time-segmented method switched from MS/ MS mode for ZnPPIX detection to MS3 mode for MPIX and PPIX measurement. 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 detection with the specific MS3 method.

#### **Figure 4.**

*Fragmentation of PPIX in the ion trap. A: [M+H]+ ion of PPIX at m/z 563.3 in full scan mode. B: MS/MS spectrum of precursor ion at m/z 563.3. The loss of the ethanoic acid side chain (59 u) is dominant. C: MS<sup>3</sup> spectrum for two-step fragmentation (m/z 563.3 -> m/z 504.3). The major ions result from side chain cleavage (red arrows, for structure see Figure 3).*

#### **4.3 Chromatography**

HPLC separation of PPIX was not straightforward, because of its high lipophilic nature. Problems included low resolution on capillary C8 LC and high carry-over on endcapped C18 phase. The low flow rate of capillary LC (5 μl/min) was also disadvantageous for porphyrin separation. Analytical LC with its higher flow rate (300 μl/min) in conjunction with a semi-porous C18 column media and an almost isocratic gradient allowed much more efficient operation at great resolution. **Figure 5** shows the separation of a standard solution of ZnPPIX (10 pmol on column), MPIX and PPIX (5 pmol on column each).

#### **4.4 Procedure**

All experiments were performed in accordance with the declaration of Helsinki and by approval of the Ethics Committee of the Ärztekammer Westfalen-Lippe (2017-169-f-S).

**63**

**Figure 5.**

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

PPIX was obtained from Enzo life sciences GmbH (Lörrach, Germany), MPIX

*Extracted ion chromatogram (EIC) of porphyrin standard (ZnPPIX m/z 565.2; MPIX m/z 449.3; PPIX m/z 445.3)* 

For drying of samples, a SpeedVac system (Savant SPD 111V SpeedVac concentrator with vapor trap Savant RVT 5105) was used (Thermo Fisher Scientific, Schwerte, Germany). For centrifugation, a Universal 320R Hettich centrifuge

Hemolysis of 200 μl whole blood or serum was performed by adding 800 μl of water and shaking at room temperature for 1 h. Protein precipitation and porphyrin extraction was achieved by adding 2 ml of ACN, shaking at room temperature for 1 h and centrifuging at 14.000 rcf at 20 °C for 30 min. The clear supernatant was then transferred to an SPE cartridge, which was treated as described in **Table 1**. The 1st and 2nd eluates were dried and finally reconstituted in 15 μl DMSO for

Raw data were converted using the msConvert toolkit from ProteoWizard software (version 3; [53]). MPIX and PPIX were quantified using the total areas for

matogram (EIC) *m*/*z* 449.3, 479.3, 493.3; PPIX: *m*/*z* 445.3, 459.3, 489.3) and Skyline

mode (MPIX: extracted ion chro-

and ZnPPIX from Merck KGaA (Darmstadt, Germany). ACN, water, FA and methanol (MeOH) were all LC-MS grade and purchased from Merck KGaA as were ammonium hydroxide solution and dimethyl sulfoxide (DMSO). SPE cartridges

*as detected with a time-segmented method on the Esquire 3000 ion trap (for parameters see 4.4).*

came from Restek GmbH (Bad Homburg, Germany).

(Tuttlingen, Germany) was utilized.

HPLC-MS (for parameters see **Tables 2** and **3**).

the three most abundant fragment ions in MS3

software (version 20.1; [54]).

*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 5.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

**62**

**4.4 Procedure**

(2017-169-f-S).

**4.3 Chromatography**

*(red arrows, for structure see Figure 3).*

*Fragmentation of PPIX in the ion trap. A: [M+H]+*

**Figure 4.**

column), MPIX and PPIX (5 pmol on column each).

HPLC separation of PPIX was not straightforward, because of its high lipophilic nature. Problems included low resolution on capillary C8 LC and high carry-over on endcapped C18 phase. The low flow rate of capillary LC (5 μl/min) was also disadvantageous for porphyrin separation. Analytical LC with its higher flow rate (300 μl/min) in conjunction with a semi-porous C18 column media and an almost isocratic gradient allowed much more efficient operation at great resolution. **Figure 5** shows the separation of a standard solution of ZnPPIX (10 pmol on

*spectrum of precursor ion at m/z 563.3. The loss of the ethanoic acid side chain (59 u) is dominant. C: MS<sup>3</sup> spectrum for two-step fragmentation (m/z 563.3 -> m/z 504.3). The major ions result from side chain cleavage* 

 *ion of PPIX at m/z 563.3 in full scan mode. B: MS/MS* 

All experiments were performed in accordance with the declaration of Helsinki and by approval of the Ethics Committee of the Ärztekammer Westfalen-Lippe

*Extracted ion chromatogram (EIC) of porphyrin standard (ZnPPIX m/z 565.2; MPIX m/z 449.3; PPIX m/z 445.3) as detected with a time-segmented method on the Esquire 3000 ion trap (for parameters see 4.4).*

PPIX was obtained from Enzo life sciences GmbH (Lörrach, Germany), MPIX and ZnPPIX from Merck KGaA (Darmstadt, Germany). ACN, water, FA and methanol (MeOH) were all LC-MS grade and purchased from Merck KGaA as were ammonium hydroxide solution and dimethyl sulfoxide (DMSO). SPE cartridges came from Restek GmbH (Bad Homburg, Germany).

For drying of samples, a SpeedVac system (Savant SPD 111V SpeedVac concentrator with vapor trap Savant RVT 5105) was used (Thermo Fisher Scientific, Schwerte, Germany). For centrifugation, a Universal 320R Hettich centrifuge (Tuttlingen, Germany) was utilized.

Hemolysis of 200 μl whole blood or serum was performed by adding 800 μl of water and shaking at room temperature for 1 h. Protein precipitation and porphyrin extraction was achieved by adding 2 ml of ACN, shaking at room temperature for 1 h and centrifuging at 14.000 rcf at 20 °C for 30 min. The clear supernatant was then transferred to an SPE cartridge, which was treated as described in **Table 1**. The 1st and 2nd eluates were dried and finally reconstituted in 15 μl DMSO for HPLC-MS (for parameters see **Tables 2** and **3**).

Raw data were converted using the msConvert toolkit from ProteoWizard software (version 3; [53]). MPIX and PPIX were quantified using the total areas for the three most abundant fragment ions in MS3 mode (MPIX: extracted ion chromatogram (EIC) *m*/*z* 449.3, 479.3, 493.3; PPIX: *m*/*z* 445.3, 459.3, 489.3) and Skyline software (version 20.1; [54]).

### *Mass Spectrometry in Life Sciences and Clinical Laboratory*


#### **Table 1.**

*Protocol for the purification of neutral porphyrin extract with SPE cartridges.*


#### **Table 2.**

*HPLC-MS parameter for porphyrin detection.*


**65**

**Figure 7.**

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

Calibration was performed in the relevant matrix (whole blood of a healthy

*Calibration curve for PPIX spiked into the whole blood of a healthy volunteer. MPIX (IS) was spiked at* 

Samples from a patient harboring a GBM and undergoing surgery were collected

**Figure 7** shows the results. The level prior to ALA administration was measured twice (two different extractions from the same blood sample) and was 33.9 ±0.4 and 34.9 ± 1.9 pmol PPIX/ml whole blood. After ALA administration, the PPIX level rose about four-fold to 100.9 ± 2.7 and 114.4 ± 11.4 pmol PPIX/ml, respectively, at

*Overlay of three EIC traces (MPIX m/z 449.3; PPIX m/z 445.3) from measurement of whole blood from a GBM patient taken at different time points. The red line shows a sample prior to ALA administration, whereas* 

*the blue and black lines mark the samples 5 and 7 h after ALA administration.*

at different time points before, during and after surgery. The first sample was obtained 1 h prior to ALA administration for the determination of the basic free PPIX level, the following samples were collected 5 and 7 h after ALA administration. Whole blood samples were refrigerated and stored in the dark. Porphyrins were

extracted in triplicate using 200 μl of whole blood for each experiment.

volunteer) using 500 fmol to 50 pmol PPIX spikes and 500 fmol/μl MPIX (**Figure 6**). The contribution of native PPIX was determined in an aliquot spiked

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

with MPIX only.

**Figure 6.**

**5. Measurement of clinical samples**

*500 fmol/*μ*l. Five replicate injections (10* μ*l each) were run.*

#### **Table 3.**

*Parameters for MS/MS detection of ZnPPIX and MS3 detection of PPIX and MPIX.* *Protoporphyrin IX Analysis from Blood and Serum in the Context of Neurosurgery… DOI: http://dx.doi.org/10.5772/intechopen.95042*

#### **Figure 6.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

Sample load porphyrin extract (~3 ml)

1st Elution 2 ml ACN with 2% FA 2nd Elution 2 ml ACN with 20% FA

*Protocol for the purification of neutral porphyrin extract with SPE cartridges.*

1st Wash 2 ml 5% ammonium hydroxide solution

**Step Solvent** Cartridge conditioning 2 ml ACN Equilibration 2 ml water

2nd Wash 2 ml MeOH

Column Poroshell C18 (2.7 μm, 2.1 mm i.d.) with guard column

Gradient t [min] solvent B [%]

**Analyte Time [min] Mode Isolation** *m***/***z* **Fragmentation amplitude**

MS3 508.3 ± 2.0 0.70

MS3 504.3 ± 2.0 0.70

 *detection of PPIX and MPIX.*

ZnPPIX 3.4–4.3 MS/MS 625.2 ± 5.0 0.43 MPIX 4.3–5.3 MS/MS 567.3 ± 2.0 0.90

PPIX 5.3–7.0 MS/MS 563.3 ± 2.0 0.85

0.0 70 1.0 100 5.0 100 5.5 70 10.0 70

Flow 0.3 ml/min

Injection volume 10 μl **MS-Parameter** ESI (+) Capillary −4.5 kV End plate −500 V Nebulizer 30.0 psi Dry gas 9 l/min Dry temperature 320°C Scan range 400–700 *m*/*z*

Solvent A 95% water/4.9% ACN/0.1% FA Solvent B 95% ACN/4.9% water/0.1% FA

**LC-Parameter**

**Table 1.**

**64**

**Table 3.**

**Table 2.**

*HPLC-MS parameter for porphyrin detection.*

*Parameters for MS/MS detection of ZnPPIX and MS3*

*Calibration curve for PPIX spiked into the whole blood of a healthy volunteer. MPIX (IS) was spiked at 500 fmol/*μ*l. Five replicate injections (10* μ*l each) were run.*

Calibration was performed in the relevant matrix (whole blood of a healthy volunteer) using 500 fmol to 50 pmol PPIX spikes and 500 fmol/μl MPIX (**Figure 6**). The contribution of native PPIX was determined in an aliquot spiked with MPIX only.

### **5. Measurement of clinical samples**

Samples from a patient harboring a GBM and undergoing surgery were collected at different time points before, during and after surgery. The first sample was obtained 1 h prior to ALA administration for the determination of the basic free PPIX level, the following samples were collected 5 and 7 h after ALA administration. Whole blood samples were refrigerated and stored in the dark. Porphyrins were extracted in triplicate using 200 μl of whole blood for each experiment.

**Figure 7** shows the results. The level prior to ALA administration was measured twice (two different extractions from the same blood sample) and was 33.9 ±0.4 and 34.9 ± 1.9 pmol PPIX/ml whole blood. After ALA administration, the PPIX level rose about four-fold to 100.9 ± 2.7 and 114.4 ± 11.4 pmol PPIX/ml, respectively, at

#### **Figure 7.**

*Overlay of three EIC traces (MPIX m/z 449.3; PPIX m/z 445.3) from measurement of whole blood from a GBM patient taken at different time points. The red line shows a sample prior to ALA administration, whereas the blue and black lines mark the samples 5 and 7 h after ALA administration.*

#### **Figure 8.**

*Time series of a GBM patient with ALA administration at 9 am, illustrated by blue bar and blue points. The pre-ALA PPIX level was confirmed in the same blood sample (orange triangle). PPIX levels in blood of healthy volunteers were determined for comparison (green crosses).*

the later time points. For comparison, PPIX levels were determined in two healthy volunteers without ALA administration. They reached only about a third of the pre-surgery level of the patient (11.0 ± 0.7 and 12.1 ± 0.4 pmol PPIX/ml). These observations are summarized in **Figure 8**. So far, having tested one patient only, the results support the hypotheses of elevated PPIX in the circulation as discussed in the literature for other types of cancer [8–11, 32] and the timely and dramatic increase after ALA administration. An extended study involving more probands is planned.

### **6. Conclusion**

MS has the huge advantage over fluorescence-based porphyrin detection that it can pinpoint the individual molecules by their mass and, adding to the specificity, by their fragmentation pattern, which can be generated in the mass spectrometer. Ambiguities as known from fluorescence spectroscopy due to varying or overlapping absorbance maxima and extinction coefficients or fluorescing matrix interferences do not occur; background corrections with complex spectral fitting algorithms are not necessary.

A LC-MS method for the quantification of metal-free PPIX in whole blood was developed. It is short (10 min) and robust (analytical LC, ion trap MS) and provides the necessary specificity and sensitivity. ZnPPIX, MPIX and PPIX can be baseline resolved without the carryover problems observed earlier. The LLE sample preparation provides high extract purity with good recovery. Importantly, ZnPPIX and PPIX can be properly distinguished during SPE clean-up. Matrix effects which would negatively affect HPLC-MS analysis were not observed.

The method is applicable to serum in the same manner, however, serum and plasma PPIX levels are much lower than those of whole blood. Unfortunately, the values reported in the literature lack confidence (**Table 4**). There is no reference range for PPIX in serum or whole blood for healthy individuals. PPIX plasma concentrations in healthy subjects after ALA administration were low and erratic, ranging from below the limit of quantification to hundreds of nmol/l [32, 55, 56]. Often, PPIX was not detected in plasma samples at all [55]. In serum, PPIX levels are lower still, even after ALA administration.

Our results indicate the same extract purity for spiked PPIX extracted from serum as from whole blood. The recovery of PPIX was even slightly better.

**67**

**Author details**

Anna Walke1,2, Eric Suero Molina2

University of Münster, Münster, Germany

provided the original work is properly cited.

The authors declare no conflict of interest.

, Walter Stummer2

2 Department of Neurosurgery, University Hospital Münster, Münster, Germany

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Core Unit Proteomics, Interdisciplinary Center for Clinical Research,

\*Address all correspondence to: koenigs@uni-muenster.de

and Simone König1

\*

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

Nevertheless, quantification requires more effort, because only trace amounts of

Protoporphyria 2-15 [56] Healthy adults after ALA administration 0.2-2.8 [32]

**PPIX in plasma (HPLC-FLD) [nmol/l] Reference**

17.8–444.3 [55]

In proof-of-principle experiments the LC-MS method was applied to blood samples from a GBM patient, which confirmed both the elevated PPIX levels in the blood of GBM patients and the increase following ALA administration. The method is now ready for patient screening. Additional resolution and sensitivity for high throughput analysis could be achieved with instrumentation such as a triple quad-

The authors thank Doreen Ackermann for technical and Carl Zeiss Meditec for financial support. The publication was supported by the Open Access Fonds of the

endogenous PPIX were detected in sera of healthy adults so far.

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

*Reported PPIX levels in plasma, measured with HPLC-FLD.*

rupole mass spectrometer, if desired.

**Acknowledgements**

**Table 4.**

University of Münster.

**Conflict of interest**

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


### **Table 4.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

*volunteers were determined for comparison (green crosses).*

the later time points. For comparison, PPIX levels were determined in two healthy volunteers without ALA administration. They reached only about a third of the pre-surgery level of the patient (11.0 ± 0.7 and 12.1 ± 0.4 pmol PPIX/ml). These observations are summarized in **Figure 8**. So far, having tested one patient only, the results support the hypotheses of elevated PPIX in the circulation as discussed in the literature for other types of cancer [8–11, 32] and the timely and dramatic increase after ALA administration. An extended study involving more probands is planned.

*Time series of a GBM patient with ALA administration at 9 am, illustrated by blue bar and blue points. The pre-ALA PPIX level was confirmed in the same blood sample (orange triangle). PPIX levels in blood of healthy* 

MS has the huge advantage over fluorescence-based porphyrin detection that it can pinpoint the individual molecules by their mass and, adding to the specificity, by their fragmentation pattern, which can be generated in the mass spectrometer. Ambiguities as known from fluorescence spectroscopy due to varying or overlapping absorbance maxima and extinction coefficients or fluorescing matrix interferences do not occur; background corrections with complex spectral fitting

A LC-MS method for the quantification of metal-free PPIX in whole blood was developed. It is short (10 min) and robust (analytical LC, ion trap MS) and provides the necessary specificity and sensitivity. ZnPPIX, MPIX and PPIX can be baseline resolved without the carryover problems observed earlier. The LLE sample preparation provides high extract purity with good recovery. Importantly, ZnPPIX and PPIX can be properly distinguished during SPE clean-up. Matrix effects which

The method is applicable to serum in the same manner, however, serum and plasma PPIX levels are much lower than those of whole blood. Unfortunately, the values reported in the literature lack confidence (**Table 4**). There is no reference range for PPIX in serum or whole blood for healthy individuals. PPIX plasma concentrations in healthy subjects after ALA administration were low and erratic, ranging from below the limit of quantification to hundreds of nmol/l [32, 55, 56]. Often, PPIX was not detected in plasma samples at all [55]. In serum, PPIX levels

Our results indicate the same extract purity for spiked PPIX extracted from

serum as from whole blood. The recovery of PPIX was even slightly better.

would negatively affect HPLC-MS analysis were not observed.

are lower still, even after ALA administration.

**66**

**6. Conclusion**

**Figure 8.**

algorithms are not necessary.

*Reported PPIX levels in plasma, measured with HPLC-FLD.*

Nevertheless, quantification requires more effort, because only trace amounts of endogenous PPIX were detected in sera of healthy adults so far.

In proof-of-principle experiments the LC-MS method was applied to blood samples from a GBM patient, which confirmed both the elevated PPIX levels in the blood of GBM patients and the increase following ALA administration. The method is now ready for patient screening. Additional resolution and sensitivity for high throughput analysis could be achieved with instrumentation such as a triple quadrupole mass spectrometer, if desired.
