Development and validation of a capillary electrophoresis tandem mass spectrometry analytical method for the determination of Leu-Val-Val- and Val-Val-hemorphin-7 peptides in cerebrospinal fluid

Journal of Chromatography A, 1267 (2012) 170–177

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Development and validation of a capillary electrophoresis tandem mass spectrometry analytical method for the determination of Leu-Val-Val- and Val-Val-hemorphin-7 peptides in cerebrospinal fluid Mariangela Zeccola a , Renato Longhi b , Diana Valeria Rossetti c , Luca D’Angelo c,d , Gianpiero Tamburrini d , Concezio Di Rocco d , Bruno Giardina a,c , Massimo Castagnola a,c , Claudia Desiderio a,∗ a Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, L.go F. Vito 1, 00168 Rome, Italy b Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milan, Italy c Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, L.go F. Vito 1, 00168 Rome, Italy d Reparto di Neurochirurgia Infantile, Istituto di Neurochirurgia – Policlinico Universitario A. Gemelli, Rome, Italy

a r t i c l e

i n f o

Article history: Available online 14 July 2012 Keywords: Capillary electrophoresis Mass spectrometry Hemorphins Cerebrospinal fluid

a b s t r a c t A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15 min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid. © 2012 Elsevier B.V. All rights reserved.

1. Introduction LVV- and VV-hemorphin-7 (LVV- and VV-h7) are members of the hemorphins family, a group of non-classical opioid peptides with affinity towards ␮- and ␴-opioid receptors [1,2]. Based on the N-terminal sequence, the following hemorphins sub-families can be distinguished: LVV-, VV-, V-hemorphins and hemorphins [2]. The sequence YPW is essential for the opioid activity in the binding capacity to the opiate receptor. LVVand VV-hemorphin-7 are deca- and nona-peptides, respectively, with sequence corresponding to fragment 32–41 and 33–41, respectively, of the haemoglobin beta chain (Table 1). In fact,

∗ Corresponding author at: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare – Sezione di Roma, c/o Istituto di Biochimica e Biochimica Clinica, dell’Universita’ Cattolica del S. Cuore, L.go F. Vito 1, 00168 Rome, Italy. Tel.: +39 06 3053598; fax: +39 06 3057612. E-mail address: [email protected] (C. Desiderio). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.07.007

the hemorphins are endogenous bioactive peptides derived from haemoglobin through the action of tissue specific proteolytic enzymes [2–4]. LVV- and VV-h7 were usually found in the central nervous system (CNS) tissues [1,4,5]. They are probably generated by cathepsin D [6,7]. Although, it was initially supposed that the source of hemorphins was erythrocyte haemoglobin (Hb), a key paper demonstrated that the brain Hb derived fragmentome origins only minimally from erythrocyte haemoglobin [8]. Rather, alpha and beta globin chains expressed in neurons and glial cells of rat and human could be the main source of brain hemorphins [9,10]. In addition to opioid like action, other relevant biological activities of hemorphins have been discovered: the capacity of lowering blood pressure, as inhibitors of angiotensin converting enzyme (ACE), their connection to beta-endorphin release and the low affinity towards orphan bombesin receptor subtype 3, involved in the modulation of intracellular calcium and protein phosphorylation [1,11]. Particularly, the LVV-h7 was supposed to be the endogenous ligand of the At4 receptor, the same receptor of angiotensin IV that is widely distributed in brain and peripheral tissues [12].

M. Zeccola et al. / J. Chromatogr. A 1267 (2012) 170–177 Table 1 Characteristics of analysed peptides. Amino acid sequence

Protein reference

Mr

LVVYPWTQRF VVYPWTQRF

Hb (human) subunit ␤ fragment 32–41 Hb (human) subunit ␤ fragment 33–41

1308.4 1194.3

Recently, the important role of hemorphins in the organism resistance to homeostatic disturbance was also reported [13]. Due to the interesting biological activity of hemorphins, several papers studied their presence and variation in tissue and biological fluids in relation to different diseases. LVV-h7 was found in ventricular cerebrospinal fluid of patients with intracerebral bleeding but not in spinal samples of healthy subjects [14]. The ligand capacity of hemorphins to At4 receptor and the inhibition of ACE stimulated the investigation of their possible role in neurodegenerative diseases [15] and the study of their possible administration as therapeutic drug [16]. A general increase of all hemorphins, including LVV-hemorphin-7, was recognised in brain tissues from Alzheimer’s disease patients, probably evidencing a vascular abnormality by amyloid angiopathy of the disease [15]. In another study the central administration of LVV-h7 improved learning and memory in normal rodents and was able to reverse memory deficits in animal models with amnesia [16]. LVV- and VV-hemorphin-7 were identified in both adrenal tissues of patient affected by pheochromocytoma tumour and healthy subjects [17]. LVV-h7 was found in an isolated BAL sample of lung cancer patient [18]. Reduced levels of hemorphins-7 were present in serum of breast cancer patients with respect to controls, suggesting their role as disease biomarker [19]. In vitro studies on tumour cell cultures evidenced a cytotoxic and antiproliferative activity of different hemorphin groups [20]. Particularly, the LVV-type resulted more antiproliferative than cytotoxic, whereas the VV-type and the valorphin, corresponding to the sequence VVYPWTQ, exhibited the reverse effects. The presence of Leu at the N-terminal produces the loss of cytotoxicity for hemorphin whereas in VV-hemorphins the presence of Gln is essential for cytotoxic effects. Recent proteomic studies of our group evidenced a potential biomarker role of LVV- and VV-hemorphins-7 in paediatric posterior cranial fossa brain tumour [21]. The two peptides were present only in the post-surgery cerebrospinal fluid after total tumour removal and in the control samples of age in the range of patients and are good prognostic candidates. In fact a partial tumour resection or the presence of metastasis in the patient at the diagnosis did not restore the hemorphins content in post-surgery cerebrospinal fluid. These results stimulated the development of a validated analytical method allowing fast screening of LVV- and VV-h7 in multiple samples characterised by precision, accuracy and fast analysis time for deeper investigations and possible future diagnostic applications. The first analytical method for the determination of LVV-h7 in ventricular cerebrospinal fluid of patients with intracerebral bleeding was carried out by size exclusion chromatography and ESI mass spectrometry [14]. In recent years, together with HPLC analytical technique and mass spectrometry or UV detection, hemorphins determinations in biological matrix were also performed using capillary electrophoresis (CE) technique [21–23] with UV and/or off-line MS detection. In the view of administration of hemorphins as drug, a CZE-UV method was developed for the evaluation of LVV-hemorphin-7 catabolism induced by aminopeptidase M or ACE enzymes cleavage in spiked human plasma [21] and in vitro [23,24]. Off-line MALDI-TOF was used for metabolites characterisation. No papers report in literature the development of a CE

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analytical method in coupling with on-line ESI mass spectrometry for hemorphins quantitation. CE and CE-ESI-MS techniques were successfully applied to proteins and peptides analysis, as documented in recent review papers [25–27]. The direct coupling of CE with mass spectrometry provides immediate specificity to CE analytes quantification well matching the analytical power of the technique with the selectivity and structure characterisation of tandem mass spectrometry detection. The purpose of our work was the optimisation and validation of a new analytical method by capillary electrophoresis in coupling with on-line ESI-mass spectrometry for the dosage of LVV- and VV-h7 in cerebrospinal fluid using the corresponding deuterated peptides as internal standard. The method was optimised by studying the most critical parameters influencing both the CE separation and the peptides mass spectrometry ionisation, and validated by testing linearity, precision, accuracy and recovery. 2. Materials and methods 2.1. Chemicals Methanol and acetonitrile LC–MS, trifluoroacetic acid (TFA) (HPLC) and formic acid (98%) were from Mallinckrodt Baker B.V. (Deventer, The Netherlands). Ultra pure water was obtained from P.Nix Power System Apparatus, Human, Seoul, Korea. Sodium hydroxide pellets, pro analysis, were from Merck, Darmstadt, Germany. LVV-, VV-hemorphin-7 and the corresponding deuterated (d8) peptides were synthesised in laboratory following the procedure described in Section 2.3. The LVV- and VV-hemorphins stock solutions 2 × 10−3 M were prepared by dissolving the synthesised peptides hydrolysate with pure methanol and stored at −20 ◦ C. Further dilutions were made in 0.1% (v/v) TFA aqueous solution and stored until use at −20 ◦ C. 2.2. Apparatus Capillary electrophoresis automated apparatus was from Agilent Technologies (Waldbronn, Germany) equipped with diode array UV detector and external nitrogen pressure. The CE apparatus was coupled to the Esquire 3000 plus mass spectrometer (Bruker Daltonics, Bremen, Germany) via a coaxial sheath liquid electrospray ionisation (ESI) interface (Agilent Technologies, Waldbronn, Germany). The sheath liquid was delivered by an external syringe pump (Cole Palmer, Vernon Hills, IL, USA) at a constant flow rate of 180 and 240 ␮L/h for CE-MS and flow injection analysis (FIA), respectively. Nebulising and drying gas (nitrogen) were set at 41368.5 Pa and 4.0 L/min, respectively. Dry gas temperature was 250 ◦ C. Mass spectrometry capillary voltage was 4500 V. Separations were performed in 50 ␮m I.D., 375 ␮m O.D. fused silica uncoated capillaries (Composite Metal Services, Hallow, Worcs., UK) of total length of 87 cm. Effective length was 21.5 cm for UV detection and 87 cm for MS detection. Tandem mass spectrometry (MS2 ) detection of the analytes was performed in product ion scan mode by activating the Multiple Reaction Monitoring (MRM) windows using an isolation width of ±4.0 m/z and fragmentation amplitude of 1.0 V and 2.0 V for the hemorphins and d8-hemorphins, respectively, in positive ionisation mode and normal resolution scan. The acquisition of the MS2 extracted ion current (EIC) signals was made in 400–1400 m/z mass scan range using a maximum accumulation time of 200 ms and a set target value of 50,000 and by activating the ion charge control (ICC) function. The sheath liquid consisted of water/methanol

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30:70 (v/v) mixture containing 0.1% (v/v) formic acid (final concentration). The temperature of the CE-MS assembly cartridge was set at 25 ◦ C. The CE running voltage was 27 kV (positive polarity). Samples were injected at the anodic end at 50 mbar × 10 s followed by BGE injection at 50 mbar × 15 s. New capillaries were conditioned using the following procedure: (1) water (5 bar × 2 min); (2) 0.1 M sodium hydroxide (4 bar × 20 min); (3) water (5 bar × 5 min). At the beginning and at the end of each working day, the capillary was rinsed with water (6 bar × 2 min), 0.1 M sodium hydroxide (6 bar × 2 min); (3) and water (6 bar × 3 min). Between runs washings were performed using both a pre- and post-conditioning as following specified. Pre-run conditioning was performed by water (6 bar × 1 min) and BGE (6 bar × 1.5 min). Post-run conditioning was made by 7.5% (v/v) formic acid in water/acetonitrile 20:80 (v/v) solution (5 bar × 1 min) followed by 7.5% (v/v) formic acid aqueous solution (6 bar × 1.2 min) and water (6 bar × 1.5 min). The post-run conditioning ensured the immediate cleaning of the capillary from biological materials after each run. The BGE and the sheath liquid were daily prepared. The BGE consisted of formic acid aqueous solution. The sheath liquid, consisting of water/methanol solution 30:70 (v/v), was sonicated for 15 min at room temperature, and successively added of 0.1% (v/v) formic acid before use.

2.4. Cerebrospinal fluid samples An aliquot of the cerebrospinal fluid (CSF) routinely collected from paediatric patients for chemical and microbiological analysis and removed to control intracranial pressure (ICP) during the surgical operation and the immediate postoperative course was utilised for the present study with the written consent of children parents. CSFs were obtained from children affected by posterior cranial fossa tumours at diagnosis admitted to the Pediatric Neurosurgery Unit of Catholic University of Rome. The CSFs collected from patients affected by congenital non-tumoural obstructive hydrocephalus were the control samples. Pre-operative CSF (PRE-CSF) samples were acquired intraoperatively before the tumour removal. Post-operative CSF (POST-CSF) samples were acquired six days after the surgery before the removal of the ventricular catheter or during surgery in case of surgical treatment of the persistent hydrocephalus. Ctrl patients underwent endoscopic third-ventriculostomy (ETV) for the treatment of the congenital hydrocephalus and the CSF was collected during the surgery. All the CSF samples were collected under sterile conditions and immediately stored at −80 ◦ C. After thawing 40 ␮L aliquot of CSF samples was diluted 1:1 with 0.2% (v/v) TFA aqueous solution, vortex mixed and centrifuged (10 min at 21,952 × g, 4 ◦ C). A volume of 20 ␮L of the acidic fraction (supernatant) was introduced into the CE-MS apparatus for LVV- and VV-hemorphin-7 analysis.

2.3. Synthesis of peptides Peptides were assembled on an Applied Biosystem Peptide Synthesizer 433A (Foster City, CA, USA). N-␣-Fmoc-l-Phenylalanine and l-phenyl-d5 -alanine-2,3,3-d3 -N-Fmoc (from CDM Isotopes, Canada) were anchored to 2-chlorotrityl chloride resin (Novabiochem, Laufelfingen, CH) according with the procedure described by Barlos et al. [28]. Peptides were synthesised following the Fmoc-(N˛ -9-Fluorenyl-methyloxycarbonyl) protocol for stepwise solid phase peptide synthesis [29,30]. Fmoc-amino acids and activators were from Novabiochem. All couplings were carried out with 5-fold excess of activated amino acid in the presence of 10 equiv. of Nethyldiisopropyl amine, using 5 equiv. of 2-(1H-benzotriazole-1yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate as activating agent for the carboxy group. At the end of peptide chain assembly, the peptide was cleaved from the resin by treatment with a mixture of (v/v) 80% trifluoroacetic acid, 5% water, 5% phenol, 5% thioanisole, 2.5% 2,2 -(ethylenedioxy)di-ethanthiol and 2.5% triisopropylsilane for 3 h at room temperature, with concomitant side chain deprotection. The resin was filtered and the peptide was precipitated in cold tert-butylmethyl ether. After centrifugation and washing with tert-butylmethyl ether the peptide was suspended in 5% (v/v) aqueous acetic acid and lyophilised. Analytical and semipreparative Reversed Phase High Performance Liquid Chromatography (RP-HPLC) was carried out on a Tri Rotar-VI HPLC system equipped with a MD-910 multichannel detector for analytical purposes or with a Uvidec-100-VI variable UV detector for preparative purpose (all from JASCO, Tokyo, Japan). Analytical RP-HPLC was performed on a Jupiter 4␮ Proteo 90A column (150 mm × 4.6 mm, Phenomenex, Torrance, CA, USA). Semipreparative RP-HPLC was performed using a Jupiter 10␮ Proteo 90A (250 mm × 21.2 mm, Phenomenex, Torrance, CA, USA). Linear gradients of acetonitrile in aqueous 0.1% TFA (v/v) were used to elute bound peptide. Peptide was recovered, spectrophotometrically quantitated and lyophilised. MALDI-TOF mass spectrometry analyses were performed on an Autoflex workstation (Bruker Daltonics, Bremen, DE). Observed experimental values for peptide masses were in agreement with theoretical calculated values.

2.5. Preparation of calibrating and spiking standard solutions The LVV- and VV-hemorphins stock solutions were diluted with 0.1% (v/v) TFA aqueous solutions in order to prepare different calibrating standard solutions in concentration range of 0.25–16.00 ␮M (n = 9) containing a fixed internal standards final concentration (2.5 ␮M, each d8-peptide). For the preparation of the CSF calibrating samples, different peptides spiking standard solutions (n = 9) were prepared in the range of concentrations 25–160 ␮M, ten times higher than the final spiked value, in order to operate the same CSF dilution at all the concentration tested for the validation, i.e. by adding the same aliquot to all samples. These peptides mixtures did not contain the d8 peptides. A ten times concentrated mixture (25 ␮M) of the d8hemorphins was separately prepared and added to the CSF at a fixed concentration of 2.5 ␮M.

2.6. Preparation of CSF calibrating samples A pool of patient PRE-CSF was used as blank matrix for the preparation of the CSF calibrating samples used for performing the method validation on the biological matrix. Before the addition of the peptides standard mixture the pool was verified for the absence of endogenous hemorphins (see Section 3). Calibrating samples were prepared by spiking an aliquot of 30 ␮L of PRE-CSF pool with 10% volume (i.e. 3 ␮L) of the spiking standard solution (ten times concentrated, 10×) of LVV- and VVhemorphin-7 and 10% volume (i.e., 3 ␮L) of the d8-peptides mixture in order to have six different samples in the concentration range 0.25–16 ␮M containing a fixed content of d8 peptides (internal standard, I.S.) at 2.5 ␮M. The blank (unspiked CSF) and the zero (CSF spiked with d8peptides only) samples were prepared by substituting the added volumes of spiking and I.S. concentrated solutions with 0.1% (v/v) TFA aqueous solution.

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Fig. 1. Full scan MS spectra of LVV- and VV-hemorphin-7 and relative d8-peptides standard compounds obtained by flow injection analysis (FIA). Sheath liquid: water/methanol (30:70, v/v), 0.1% (v/v) formic acid at flow rate 240 ␮L/h. Other experimental conditions are described in Section 2.

2.7. Method validation

3.1. Optimisation of MS ionisation parameter

The CSF calibrating samples were prepared in triplicate to test the linearity of the method. Three different calibration curves were performed in the range of concentration 0.25–16 ␮M and the regression equation from the average values were used for calculation of method precision and accuracy. The CSF calibrating samples (quality control samples, QCs) at 2, 4 and 12 ␮M, corresponding to a low (LLOQ), medium (mQCs) and high concentration (hQCs) samples, were prepared in quintuplicate and tested for evaluation of method intra- (n = 6 consecutive runs) and inter-assay (n = 5 days, average values of three runs per day) precision, accuracy and recovery. The method recovery was calculated with respect to the response of peptides standard solutions, therefore using for calculation the regression equation obtained from the standard calibration curve. The extracted ion current (EIC) plots of the main fragments produced for each peptide were used for quantitation using the peptide/IS peak area ratio data.

Before developing the LVV- and VV-hemorphin-7 CE separation, the peptides MS ionisation was carefully optimised by flow injection analysis (FIA) using CE instrument as sampler [31] and using a solution of water/methanol 30:70 (v/v) containing 0.1% (v/v) formic acid as sheath liquid. The Smart Parameters Setting (SPS) set at 1400 m/z, in accordance with the molecular mass values of the peptides of interest, automatically adjusted the optimum values of tune source, i.e., the trap drive (100.7), the Octapole RF amplitude (178.5 Vpp), lens 2 (−60 V), Capillary Exit (196.0 V). Compound stability was set at 100%. The dry gas temperature was 250 ◦ C. Nebuliser and dry gas were erogated at 41368.5 Pa and 4.00 L/min, respectively. For the optimisation of analytes ionisation the following parameters were studied: the MS voltage, the CE capillary protrusion from the ESI source (capExit) and the nebuliser gas pressure. These parameters are the most critical for obtaining both a stable spray and an efficient ionisation and are dependent each other. The capExit can be regulated by adjusting the tally marks on the microregulation screw on the ESI needle interface selecting and turning it counter clock wise (+ values). The effect of the MS capillary voltage was investigated at 4000, 4500 and 5000 V using a capExit at +2. Although all these voltages provided comparable signal intensities, 4500 V produced the best compromise between analytes signal and spray stability and was therefore selected as the optimum value to investigate the effect of the capExit at +1, +2, +3 and +4. The highest the value of capExit, the highest the capillary protrusion from the ESI source. All the tested values showed a stable spray with the exception of +4 that produced too high ESI current, not advisable for obtaining good baseline and reproducible results. The +2 produced the most intense analytes ionisation and therefore the value was selected. The nebuliser gas was applied in the range 27579.0–55158.0 Pa. Considering the analytes signal intensity and the ESI current produced, the value of 41368.5 Pa resulted the best value to apply. Fig. 1 shows the obtained full scan MS spectra of the LVV- and VV-h7 peptides and of the corresponding d8-peptides used as internal standard (I.S.). The MS spectrum showed for all the peptides both the [M+H]+ and the [M+2H]2+ molecular ions, the last exhibiting the most intense signal.

3. Results and discussion LVV- and VV-hemorphins-7 are deca- and nona-peptides differing for only one amino acid of the sequence and corresponding to the fragments 33–41 and 32–41 of the ␤ subunit of human haemoglobin (Table 1). The development of an analytical method for their quantitation by CE-MS required the optimisation of several parameters involving both the CE separation and the analytes ionisation and detection in mass spectrometry. According to the physico-chemical properties of the analytes, the choice of type and concentration of BGE, applied voltage and delivered current are the most critical parameters for CE-MS separations. On the other end the optimisation of analytes ionisation and detection in MS requires, particularly, the study of the sheath liquid composition and flow rate, the entity of the CE capillary protrusion from the ESI source (cap-exit) and the MS voltage, all concurring to the generation of a stable spray, and, in case of MS2 or MSn detection modes, the setting of the parameters for the isolation and selective identification of fragments.

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Fig. 2. CE-MS extracted ion current (EIC) plots and MS2 spectra of a mixture of LVV- and VV-hemorphin-7 standard compounds in presence of their corresponding d8-peptides (I.S.) under the optimum experimental conditions. BGE: formic acid 1.5 M. Sheath liquid flow rate at 180 ␮L/h. Other experimental conditions as described in Section 2.

Requiring the method the analysis of the peptides in a biological matrix, the MS2 mode of detection was chosen fragmenting the [M+2H]2+ ions with an isolation window of 4 m/z. The relative spectra can be observed in Fig. 2. The fragmentation amplitude was studied in the range 0.5–2.0 set instrument values. The values 1.0 and 2.0 resulted as the best values for LVV- and VV-h7 and the corresponding deuterated peptides, respectively, in Multiple Reaction Monitoring (MRM) scan mode. According to the strong molecular similarity of LVV- and VVh7, differing for only one amino acid residue of the sequence, the MS2 spectra of these peptides showed a closely related fragmentation pattern with preponderant product ions of “y” series. In this case, although MS detection is able to provide excellent selectivity, being able to extract different signal of co-migrating substances, the baseline analytical separation of LVV- and VV-h7 peptides was necessary to be optimised for obtaining their accurate quantitation.

3.2. Optimisation of CE-MS analytes separation Preliminary CE-MS analyses were performed using a solution of volatile formic acid as BGE. Under acidic conditions the peptides of interest were positively charged, in accordance with their molecular structure and isoelectric point, migrating as positively charged compounds towards the cathode. The use of concentrated acidic BGE ensured the silanol group protonation minimising cations adsorption on the fused silica uncoated capillary wall. The peptides separation was optimised on LVV- and VV-h7 peptide standard mixture using formic acid BGE at concentrations of 1.0, 1.5 and 2.0 M. All the tested BGEs allowed the baseline separation of the two peptides. Among them, only the 1.5 M concentration provided analysis repeatability on consecutive injections (n = 3) within acceptable RSD value (<15%) together with the highest analytes signal intensities. Probably, the BGE 1.0 was not able to provide a similar stacking effect and the suitable competition in capillary wall adsorption. On the other end, the 2.0 M was generating too high current, a less stable spray and a higher suppression of analytes ionisation signals. Fig. 2 shows the optimised CE-MS separation of LVV- and VVh7 standard mixture in presence of the corresponding deuterated

peptides in less than 15 min. The VV-h7 was the first migrating peptide.

3.3. Method validation on standard solution Before starting with method validation on CSF matrix, the method was tested for the linearity, accuracy and precision on calibrating standard solution mixtures of LVV- and VV-h7 in concentration range 0.25–10 ␮M (n = 7) containing a fixed I.S. concentration of 2.5 ␮M (each d8-peptide). The obtained regression equations were y = 0.3677x + 0.0498 with R2 = 0.9917 and y = 0.2935x + 0.0466 with R2 = 0.9937 for LVV- and VV-h7, respectively, confirming the linearity of the response under the conditions used. Three concentration levels, namely 0.25 (low), 2 (medium) and 8 ␮M (high) were tested for intra- (n = 5 consecutive runs) and inter-assay (n = 5 days) accuracy and precision (Table 2). As it can be observed in Table 2, differently from the medium and high levels, the peptides concentration of 0.25 ␮M did not exhibit RSD values for accuracy and precision within acceptable RSD values, although providing a quantifiable peak area and fitting the linearity of the response. This value was therefore considered the method LOD for standard peptides analysis.

3.4. Bioanalytical method validation Method validation on cerebrospinal fluid was performed following the FDA Guidance for Bioanalytical Method Validation [32]. Before staring the quantitative tests, a pool of PRE-CSF patient samples, resulted blank for hemorphins in previous investigation [21], was prepared and used as biological matrix for the preparation of the CSF calibrating samples, as specified in details in Section 2. The CSF pool was verified to not contain LVV- and VV-h7 and method specificity was evaluated by comparing the CE-MS profiles obtained from the analysis of blank, zero (i.e. blank CSF added with d8-peptides only) and spiked (i.e. blank CSF added with both d8and standard peptides) CSF samples. As it can be observed in Fig. 3 the optimised method allowed CE-MS analysis and identification of hemorphins free from matrix interferences.

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Table 2 Intra-assay and inter-assay accuracy and precision data obtained on LVV- and VV-h7 standard solutions. Analytes

Calibration level

Nominal concentration (␮M)

Intra-assay precision (n = 5 runs)

Inter-assay precision (n = 5 days)

Inter-assay accuracy (n = 5 days)

SD

RSD

SD

RSD

(value %) ±SD

RSD

LVV-h7

Low Medium High

0.25 2 8

±0.0157 ±0.0414 ±0.2825

10.79% 5.01% 9.32%

±0.0252 ±0.0646 ±0.2766

20.19% 7.38% 8.75%

81.56 ± 27.41 112.16 ± 8.78 105.79 ± 9.40

33.61% 7.83% 8.89%

VV-h7

Low Medium High

0.25 2 8

±0.0096 ±0.1135 ±0.4469

16.74% 17.20% 14.70%

±0.0199 ±0.0977 ±0.3877

23.25% 14.21% 13.32%

53.18 ± 27.13 109.11 ± 16.64 122.01 ± 16.51

51.02% 15.25% 13.53%

When analysing CSF spiked samples, it was necessary to decrease the BGE formic acid concentration from 1.5 to 1.0 M due to the too high current generated in the system and the poor data repeatability, probably caused by the high content of salts in the samples and MS ionisation interferences. Method linearity was studied in the range of concentration 0.25–16 ␮M (n = 6) performing three different calibration curves. The 0.25 ␮M concentration provided a quantifiable signal, however, due to the unacceptable data repeatability and linearity of the response, it was excluded from quantitative validation and considered the method LOD. The lower limit of quantification (LLOQ) of the method was therefore 2 ␮M matrix concentration (each peptide). The data relative to the three different calibration curves in concentration range 2–16 ␮M (n = 5) are reported in Table 3. The obtained slope values showed acceptable RSD and correlation coefficients demonstrating the linearity of method also on biological matrix. Three different concentrations, corresponding to low (2 ␮M), medium (4 ␮M) and high (12 ␮M) calibration levels, were tested for intra- and inter-assay method accuracy and precision. The results are shown in Table 4. The obtained data showed RSD values within the acceptable data. 3.5. Analysis of patient CSF The optimised method was applied to the analysis of cerebrospinal fluid of patients affected by posterior cranial fossa brain Intensity x104 3.0

A

2.0 1.0 0.0 x105 1.0

Table 3 Linearity data (CSF matrix).

B

Parameters

0.8 0.6 0.4

1.0

d8-LVV-h7

VV-h7

0.6

LVV-h7

0.4

Slope

0.3617 0.4679 0.3794 0.4030 0.0569 14.1

Intercept

Day1 Day 2 Day 3 Average SD

−0.0314 −0.4662 −0.2294 −0.2423 0.2177

−0.0314 −0.5 892 −0.2192 −0.2799 0.2838

R

Day1 Day 2 Day 3 Average SD RSD

0.9973 0.9963 0.9912 0.9949 0.0033 0.3

0.9982 0.9890 0.9951 0.9941 0.0047 0.5

d8-LVV-h7

d8-VV-h7

0.2 0.0

6

7

8

9

10

11

12

13 Time [min]

Fig. 3. Comparison of the CE-MS extracted ion current (EIC) plots of a (A) blank sample, (B) zero sample and (C) hemorphins and d8-hemorphins spiked sample of cerebrospinal fluid (PRE-CSF pool). BGE: formic acid 1.0 M. For other experimental conditions and sample pretreatment see Section 2.

VV-h7

0.4697 0.5402 0.4766 0.4955 0.0389 7.8

C

0.8

LVV-h7 Day1 Day 2 Day 3 Average SD RSD

d8-VV-h7

0.2 0.0 x10 5

tumours for quantitation of LVV- and VV-hemorphin-7. For the same patient cerebrospinal fluid was collected both during surgery before tumour resection (PRE-CSF sample) and six days after the surgery (POST-CSF sample) before the removal of ventricular catheter. Due to the very high turnover of CSF, these two samples were considered to well represent the oncologic and non-oncologic state of the same patients. CSF samples from paediatric patients affected by congenital hydrocephalus were the control samples (Ctrl samples) of reference for the non-oncologic state. In order to test the applicability of the presented method for future clinical applications, the PRE- and POST-CSF samples from patients affected by different forms of posterior cranial fossa brain tumours, i.e. medulloblastoma (3 patients), ependimoma (1 patient) and pylocitic astrocytoma (1 patient) and two Ctrl-CSFs were analysed. For each sample the average value obtained from of at least three runs was evaluated. The CSF samples followed a very simple and rapid pretreatment procedure before the CE-MS analysis, consisting of a simple dilution step with TFA solution followed by centrifugation and recovery of the surnatant, as specified in details in Section 2. A small aliquot of 40 ␮L of CSF underwent to sample pretreatment procedure and ensured numerous repeated analyses per sample injecting only few nL each run in CE-MS instrumentation. In accordance with previous findings [21], LVV- and VV-h7 were detectable only in the analysed POST-CSF samples and in the Ctrls. Both the hemorphins were under the limit of detection of the method in the PRE-CSFs. As an example, Fig. 4 shows the extracted ion current (EIC) plots of hemorphins analysis in the PREand POST-CSF of the same patient where LVV- and VV-h7 resulted present only in the POST- one. The levels of LVV- and VV-h7 in POST-CSFs in the analysed samples resulted in concentration range 3.13 ± 0.17–8.74 ± 0.54 ␮M in accordance with the selected range

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Table 4 Intra-assay and inter-assay precision and accuracy data (CSF matrix). Analyte

Calibration level

Nominal conc. (␮M)

Intra-assay precision (n = 5 runs)

Inter-assay precision (n = 5 days)

Inter-assay accuracy (n = 5 days)

Recovery (n = 5 determinations)

SD

RSD

SD

RSD

(value%) ± SD

RSD

(value%) ± SD

LVV-h7

LLOQ mQCs hQCs

2 4 12

±0.03 ±0.23 ±0.52

3.91% 12.81% 9.40%

±0.16 ±0.10 ±0.37

15.65% 5.83% 7.14%

126.70 ± 16.0 102.50 ± 5.26 91.14 ± 6.22

12.63% 5.13% 6.82%

95.10 ± 14.80 88.70 ± 5.16 85.40 ± 6.10

VV-h7

LLOQ mQCs hQCs

2 4 12

±0.05 ±0.19 ±0.60

7.44% 12.30% 11.44%

±0.11 ±0.32 ±0.47

14.17% 19.20% 10.63%

130.20 ± 11.20 121.90 ± 20.14 98.90 ± 9.90

14.17% 16.52% 10.00%

79.20 ± 11.20 96.40 ± 18.60 94.03 ± 10.00

Intens. x105

PRE-CSF

1.0 0.8 0.6

d8-LVV-h7 d8-VV-h7

0.4 0.2 0.0 5 Intens. 5 x10 1.0 0.8

6

7

8

9

10

11

12

13

14Time [min]

LVV-H7 POST-CSF d8-LVV-h7

0.6

d8-VV-h7

0.4 0.2 0.0

VV-H7 5

6

7

8

9

10

11

12

13

14Time [min]

Fig. 4. CE-MS extracted ion current (EIC) plots of a pre-surgery (PRE-CSF) and post-surgery (POST-CSF) cerebrospinal fluid samples from the same patients. Experimental conditions as in Fig. 3.

of method validation. Two samples of POST-CSF and one Ctrl-CSF showed for the VV-h7 peptide an integrable peak corresponding to a concentration below the method LLOQ. 4. Conclusions The identification of LVV- and VV-h7 peptides as potential biomarkers of posterior cranial fossa paediatric brain tumour disease [21] and the reported role of these peptides inside neurodegenerative pathologies, demand for the development of a validated quantitative analytical method, absent in literature for their accurate and precise dosage in cerebrospinal fluid on wide screening. Although hemorphins were detected in CSF by LC–MS proteomic analysis [21], we choose CE-MS analytical technique for method development due to the miniaturisation and the simplicity combined with the high selectivity of tandem MS detection for biological matrix analysis. The method save costs and time with respect to LC due to the absence of expensive chromatographic column, preconditioning times and gradient elutions and allowed several injections per sample starting from few microliters of biological fluid. In the present method the CE was coupled with ESI-IT-MS and LVV- and VV-hemorphin-7 quantitation was performed by the internal standard method using the synthetic deuterated peptides as compounds of reference for validation. The method allowed a very reduced and rapid sample pretreatment, simply consisting in biological matrix dilution with TFA solution, centrifugation and injection of surnatant, and a fast screening of numerous samples. The method was successfully validated showing linearity, accuracy and precision data within the acceptable values and was demonstrated to be applicable to the peptides quantitation in patient samples affected by different posterior cranial fossa tumour forms

confirming our previous findings [21]. In fact the analysis of patient cerebrospinal fluid confirmed the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The availability of an accurate and precise method for quantify hemorphins in cerebrospinal fluid on a wide screening is of relevant importance to confirm their ascribed potential biomarker role in posterior cranial fossa tumours, to establish their specificity in other brain tumours forms and to study their biological role inside other neurological diseases. Relatively to posterior cranial fossa brain tumours, the dosage of LVV- and VV-h7 in post-surgery CSF could have in future relevant diagnostic applications providing a methodology with reduced invasiveness for the evaluation of the successful total tumour resection and the monitoring of the patient during the follow-up by recognition of disease onset before the clinical symptoms or by supporting suspected magnetic resonance images (MRI). Acknowledgement The authors acknowledge the Federazione Gene-Policlinico A. Gemelli (Rome) for the financial support of this study. References [1] F. Nyberg, K. Sanderson, E.L. Glamsta, Biopolymers 43 (1997) 147. [2] I. Gomes, C.S. Dale, K. Casten, M.A. Geigner, F.C. Gozzo, E.S. Ferro, A.S. Heimann, L.A. Devi, AAPS J. 12 (2010) 658. [3] V. Brantl, C. Gramsch, F. Lottspeich, R. Mertz, K.H. Jaeger, A. Herz, Eur. J. Pharmacol. 125 (1986) 309. [4] V.T. Ivanov, A.A. Karelin, M.M. Philippova, I.V. Nazimov, V.Z. Pletnev, Biopolymers 43 (1997) 188. [5] A.A. Zamyatnin, Biochemistry (Moscow) 74 (2009) 201. [6] Q. Zhao, I. Garreau, F. Sannier, J.M. Piot, Biopolymers 43 (1997) 75.

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