Effective phospholipid removal from plasma samples by solid phase extraction with the use of copper (II) modified silica gel cartridges

Accepted Manuscript Title: Effective phospholipid removal from plasma samples by solid phase extraction with the use of copper (II) modified silica gel cartridges Authors: Jolanta Flieger, Małgorzata Tatarczak-Michalewska, ´ Anna Kowalska, Anna Madejska, Tomasz Sniegocki, Anna Sroka-Bartnicka, Monika Szyma´nska-Chargot PII: DOI: Reference:

S1570-0232(17)31177-7 https://doi.org/10.1016/j.jchromb.2017.10.021 CHROMB 20851

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

8-7-2017 8-9-2017 9-10-2017

Please cite this article as: Jolanta Flieger, Małgorzata Tatarczak-Michalewska, Anna ´ Kowalska, Anna Madejska, Tomasz Sniegocki, Anna Sroka-Bartnicka, Monika Szyma´nska-Chargot, Effective phospholipid removal from plasma samples by solid phase extraction with the use of copper (II) modified silica gel cartridges, Journal of Chromatography B https://doi.org/10.1016/j.jchromb.2017.10.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effective phospholipid removal from plasma samples by solid phase extraction with the use of copper (II) modified silica gel cartridges.

Jolanta Fliegera*, Małgorzata Tatarczak-Michalewskaa, Anna Kowalskaa, Anna Madejskaa, Tomasz Śniegockib, Anna Sroka-Bartnickac, Monika Szymańska-Chargotd

a

Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4A, 20-093 Lublin, Poland. E-mail

address: [email protected] b

National Veterinary Research Institute, 24-100 Puławy, Al. Partyzantów 57, Poland

c

Department of Genetics and Microbiology, Maria Curie-Sklodowska Univeristy, Akademicka 19, 20-033

Lublin, Poland d

Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290 Lublin, Poland.

Graphical abstract

ABSTRACT The new sorbent for solid phase extraction (SPE), based on silica gel modified with a copper (II), was obtained and its application for phospholipids removal from the human plasma was tested. SPE column conditioning requirements, the volume of the plasma, the composition of the elution solvent were all established. The efficacy of the removal of phospholipids was compared for different methods such as standard protein precipitation or HybridSPE Phospholipid Ultra and HybridSPE-PPT. The sample clean-up was verified by mass

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spectrometry (MS) and by monitoring of chromatograms in the region between 190 nm and 400 nm. The Fourier Transform Infrared Spectroscopy FT-IR and confocal Raman microscopy were used to evaluating the silica gel modifications and to show the structure of lipids confined in the silica pores. Keywords: Plasma samples; solid-phase extraction; modified sorbent; phospholipids.

1. Introduction Phospholipids are the main components of cell membranes and body fluids. They are one of the most troublesome components causing difficulties in the analysis of biological samples for the determination of metabolites or xenobiotics by high-performance liquid chromatography especially when coupled with tandem mass spectrometry (LC–MS). The polar nature of phospholipids, being a consequence of presence in their structure of charged functional groups: such as a negatively charged phosphate group, positive or quaternary amino groups is visible in quantitative and qualitative interferences known as matrix effects. This is manifested itself in the form of ion suppression or reinforcement of mass signal in case of phospholipids co-eluted with the designated analyte. Phospholipids can significantly shorten analytical column life. This can cause unpredictable ion suppression and poor reproducibility. Phospholipids exist in several classes. However, only phosphatidylcholines and lysophosphatidylcholines, generating the most interference in bioanalytical assays samples of serum and plasma, have been raising particular interest in rapid LC-MS/MS. They are visualized by monitoring the 184→184 mass transition corresponding to the polar phospholipid head in positive ion mode. Although interferences caused by phospholipids concern primarily ion suppression in LC–MS/MS and decrease in MS sensitivity, very

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important disadvantages are also concerned with the decrease of column lifetimes. Adverse effects of phospholipids on LC–MS/MS have increased an interest for their selective removal. To reduce the matrix effect caused by the presence of phospholipids, many methods have been developed so far. The simplest one was sample dilution with an organic solvent, currently rarely used due to unfavorable changes in the limits of quantification [1]. In the past, the LC of biological fluids was proceeded by liquid–liquid extraction (LLE) with a mixture of chloroform, methanol, and water. There exist some modern modifications of this technique instead of chloroform introducing organic solvents such as dichloromethane, butanol or methyl tert-butyl ether [2-4]. Sometimes protein precipitation (PPT) was preferred over other sample cleanup techniques, despite the fact that this method was dedicated to the reduction of protein rather than the phospholipid content. Some studies have shown that protein precipitation using acetonitrile also allows partial removal of phospholipids. Beneficial effect compared to the PPT can be provided by application of assisted liquid extraction [5]. However, only the use of volatile organic solvents of the type of dichloromethane and tertbutyl methyl removes 99% of phospholipids [3]. The most common and efficient way to remove phospholipids from the biological material is solid phase extraction (SPE) [6-10]. Commercially available cartridges utilize the possibility of preferential binding of phospholipids by nanoparticles of metals such as zirconium or titanium [11]. Undoubtedly, a combination of various techniques and strategies can lead to potentializing of efficient extraction of phospholipids. This concept was used in the creation of Hybrid SPE-PPT columns [12]. In turn, the combination of protein precipitation using acidified acetonitrile and extraction with hybrid SPE-PPT "removal plate" reduces the effect of a matrix from 34.8% to 5.1% [13]. The capacity of different products for phospholipid removal depends on the size of samples and for HybridSPE-phospholipid cartridges, it ranges from 99.5% for 100 µL of plasma to 88% for 300 µL. Despite the existence of a number of 3

techniques to remove phospholipids from biological fluids, we are still looking for simple low-cost methods, limiting consumption of organic solvents and ensuring satisfactory efficiency. On the other hand, the phospholipids of blood can be a source of knowledge as biomarkers, signifying the disease. Thus the need for methods for isolation, removal, and quantification of the phospholipids appears to be rather dire. As a result of earlier Flieger studies [14], it was found that copper (II) silicate exhibits a strong affinity for nucleotide phosphate group. This property has been utilized to prepare new SPE cartridges of silica gel by its modification with copper (II) in an ammonia solution for phospholipids removal present in human plasma samples. This study focuses on the targeted preparation of plasma samples by the different techniques such as a standard protein precipitation (PPT), commercial phospholipid-removal plates and SPE with new cartridges containing copper silicate. The Fourier Transform Infrared Spectroscopy FT-IR and confocal Raman microscopy were used to evaluating the silica gel modifications. So far, the FT-IR and Raman spectroscopy has been found to be useful for evaluation of other mesoporous materials [15, 16]. The new product was tested to selectively remove phospholipids. 2. Experimental 2.1. Materials HPLC solvents of reagent grade were obtained from Merck (Darmstadt, Germany). Plasma samples were purchased from Bioreclamation. Water was deionized and purified by ULTRAPURE Millipore Direct-Q 3UV-R (Merck). 2.2. Sample preparation 2.2.1. Plasma protein precipitation (PPT) method

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1 mL quantities of human plasma samples were mixed with 3 mL of acetonitrile (ACN) and then centrifuged at 2500 rpm for 10 min. An aliquot of supernatant (5 µL) was injected into LC-MS/MS and HPLC-DAD. 2.2.2. SPE cartridges modified with Cu(II) 86 mL of 0.008 mol L-1 ammonia solution of copper (II) acetate (Sigma-Aldrich, St.Louis, MO, USA) adjusted to pH 10-11 with ammonia (25% with a density of 0.91 g/cm3 at 20 °C) from Sigma-Aldrich (St.Louis, MO, USA) was applied onto the SPE cartridges. The modified cartridge was washed with 2 mL of deionized water and 2 mL of acetonitrile. Solid-phase extraction was carried out using Bakerbond SPE Silica Gel 500 mg/3mL cartridges (J.T.Baker) on a Baker spe-12G apparatus. All experiments were conducted at a constant temperature of 25.0 ± 1 °C. The flow rate was 1 mL/min. Bakerbond SPE Silica Gel cartridges modified with Cu (II) were activated and conditioned with 2 mL of the mixture containing acetonitrile and 0.01 mol L-1 ammonia solution in the ratio of 6:1 (v/v). 1 mL of serum deproteinized with 3 mL of acetonitrile containing 0.01 mol L-1 ammonia solution in the ratio of 6:1 (v/v) and shaken vigorously and then centrifuged (MPW Med. Instruments centrifuge - 223e) at 906.5 x g (RCF) for 10 min. Aliquots of the supernatant were loaded onto the conditioned cartridge. Elution was carried out applying full vacuum for 5 min (-30 kPa). 5 μL of the eluate was injected directly into the LC-MS/MS. The analytical procedure was described in the Polish Patents [17]. 2.2.3. Hybrid-SPE-Phospholipid ultra-cartridges Official instruction for Hybrid SPE-phospholipid ultra-cartridges has been adapted for plasma sample analysis. Cartridges were conditioned with 3 mL of the mixture containing 3% formic acid in acetonitrile. A 1000 µL volume of human plasma was transferred into polypropylene centrifuge tubes. To this aliquot, 3ml of ACN was added and the mixture was centrifuged at 5

2500 rpm for 10 min. Aliquots of the supernatant were loaded onto the cartridge. Analyte elution was carried out applying full vacuum for 5 min (-30 kPa). 5 μL of the eluate was injected directly into the HPLC column. SPE procedure was carried out using Hybrid-SPEPhospholipid ultra-cartridges (30 mg mL-1) Supelco (Belleforte, USA). 2.2.4. Hybrid-SPE-PPT cartridges A 1000 µL volume of human plasma was transferred into polypropylene centrifuge tubes. Cartridges were activated and conditioned with 3 mL of acetonitrile. To this, an aliquot of 3 mL of 1% formic acid in ACN was added and the mixture was centrifuged at 2500 rpm for 10 min. An aliquot of the supernatant was further analyzed by solid-phase extraction (SPE). Analyte elution was carried out applying full vacuum for 5 min (-30 kPa). 5 μL of the eluate was injected directly into the HPLC column. SPE procedure was carried out using HybridSPE-PPT cartridges (30 mg mL-1) Supelco (Belleforte, USA). 2.3. HPLC conditions Chromatographic experiments were performed using LaChrom HPLC Merck Hitachi (E.Merck, Darmstadt, Germany) chromatograph model equipped with a diode array detector (L-2455), column Jetstream 2 Plus thermostat (100375, Knauer). The equipment was operated using HSM software (Merck). The column (150 mm x 4.6 mm I.D.) was packed with 5-μm Zorbax Extend-C18 (pore size: 8 nm, surface area: 180 m2 g-1) Agilent Technologies (Santa Clara, CA, USA). The column was thermostated at 20.0 ± 0.1 ºC. Retention data were recorded at a mobile phase flow rate of 1 mL min-1 with online degassing using L-7612 solvent degasser. Typical injection volumes were 20 μL, corresponding to the volume of the Rheodyne injector loop. The elution was carried out in the isocratic mode by mobile phase consisting of 10% methanol in water. The mobile phase was filtered through a Nylon 66 membrane filter (0.45 μm) Whatman (Maidstone, England) by the use of a filtration

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apparatus. The detection was set at an appropriate wavelength chosen accordingly with the recorded spectrum in the range from 200 nm to 400 nm. 2.4. LC-MS/MS conditions

The LC-MS/MS system consisted of an Agilent Series 1200 HPLC system (Agilent Technologies, Germany) connected to Sciex API 5500 Qtrap mass spectrometer (Sciex, Canada). The Analyst 1.6.2 software controlled the LC-MS/MS system and processed the data. The mass spectrometer was operated on in the positive ESI mode with a capillary voltage of 5.5 kV. The temperature of desolvation was set at 500 C, nebulizer gas (N2) – 40 psi; curtain gas (N2) – 40 psi; gas 1 (air) – 35 psi; gas 2 (air) – 35 psi. The collision gas (nitrogen) pressure was set at 3.1x10e-5 torr. The multiplier was set at 2300 V. The flow rate of mobile phase was 400 μl min-1, the injection volume – 5 μl. The chromatography was performed on a Kinetex C18 column (50 mm x 2.1 mm x 2.6 μm), connected to a C18 precolumn (4 mm x 2 mm x 4 μm). The mobile phase for LC analysis consisted of two solutions: A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The mobile phase gradient program started at 95% of A, 5% A at 2 min to 3 min, then 95% of A at 3.01 min and held for 2 min. The primary method for simultaneously detecting all glycerophosphocholines by monitoring MRM transitions (without fragmentation) 184→184 for (phosphatidylcholines) and 104→104 for (lysophosphatidylcholines) on the Applied Biosystems 5500 QTRAP employed adeclustering potential, entrance potential, collision energy, and collision cell exit potential of 165, 10, 7, and 5V, respectively. The collision gas (nitrogen) pressure was set to medium (3.1x10e-5torr) [18, 19, 20]. 2.5. FT -IR spectra FT-IR spectra were collected with the use of a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA) with the Smart iTR ATR sampling accessory. Each sample

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was applied on ATR as it was obtained. The spectra were collected over the range 4000–650 cm−1. For each material, five samples under the same conditions were examined. Each spectrum resulted from 64 scans to obtain an optimal signal-to-noise ratio. Then, for a given material, a final average spectrum was calculated. Baseline corrections, if needed, were obtained using Omnic Software (v. 8.2, Thermo Fischer Scientific Inc., USA). Further analysis of spectra was performed using Origin Software (OriginLab v8.5 Pro, Northampton, USA). 2.6. Raman spectra and depth profiles The DXR Raman Microscope (Thermo Scientific, Waltham, USA) was used for obtaining the Raman spectra and depth profiles imaging. The system is equipped with a diode-pumped, solid-state (DPSS) green laser ( = 532 nm) with a maximum power of 10.0 mW, a diffraction grating of 900 lines per mm and a pinhole confocal aperture of 25 µm. The Raman light was detected with an air-cooled CCD detector with a spectral resolution of 4 cm-1. The 20x/0.40NA objective was used. The samples in form as were obtained were placed on the microscopic glass slide covered by aluminum. The Raman depth profiles were recorded in point with a spatial resolution of 2 µm in the z direction. The horizontal x displacement was fixed. The scheme of the Raman depth profiles acquisition is presented in Fig. S1†. The depth profiles allow observing the changes of each component distribution along the z axis (the depth). On every depth, the Raman spectrum is obtained from each of the z/Raman shift an image is constructed. The distribution of given component is depicted as the change of intensity of characteristic for this component band along the z-axis. The integration time (30 s) was fixed for each scan. A single spectrum at each point was recorded within the range of 3500–150 cm-1 of Raman shift for an average of 12 scans. The spectra were not normalized. The Omnic software (v. 8.2, Thermo Fischer

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Scientific Inc., USA) was used for spectral smoothing and preprocessing. Further analysis of spectra was performed using Origin Software (OriginLab v 8.5 Pro, Northampton, USA).

3. Results and discussion 3.1. Copper (II) modified silica gel Initially, the influence of the modifying solution on sorption of Cu(II) was examined. In order to estimate the breakthrough volume and concentration, different volumes from 1.00 to 160 mL at the concentration range of 0.004–0.01 mol L-1 of the tetra-ammine copper (II) ions were applied on the SPE cartridges. The experiment was conducted until the whole sorbent colored into blue (Fig. S2†). As it can be seen in Fig. S3†, sorption of Cu(II) on silica gel was independent of the concentration and volume of modifying solution and remained almost at a constant level of 1.3760 x 10-3 ± 1.5650 x 10-5 moles in relation to 1 gram of silica gel. The solutions at the higher concentration of 0.01 mol L-1 proved to be disadvantageous, giving an excess of copper(II) complex collected in the eluate. As it can be noticed, only 86 mL of the modifying solution at the concentration of 0.008 M can be applied on silica gel cartridge to avoid a simultaneous retention/elution phenomenon causing waste of the tetra-ammine copper (II) ions. So, as the best modifying solution 86 mL of 0.008 M copper (II) acetate in ammonia was chosen for modification of 500 mg of silica gel in subsequent experiments. 3.2 Capacity of silica gel modified with Cu(II) (500mg Bed) Standard Protein Precipitation (control) was performed by placing 1ml of human plasma and 3 ml of acetonitrile into the tube. The tube was agitated then spun on the centrifuge to remove solids. The supernatant was analyzed directly by HPLC-DAD. Protein precipitation was compared to the efficiency of phospholipid removal by modified with Cu(II) cartridges using increasing volumes of sample. Effect of the sample size on phospholipid removal is presented

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using overlay chromatograms in Fig.1. Removal of phospholipids in percentages was calculated basing on LC-MS/MS analysis after conversion of their content into the 2-lysoglycerophosphocholine. The maximum amount of plasma was measured by passing its increasing volume from 100 µL to 18 mL through the modified extracting column. The way of operation involves loading of increasing volume of sample keeping the constant ratio 1:3 of the volume of plasma to acetonitrile. The obtained results show that up to 1 mL of plasma could be loaded onto modified cartridge ensuring almost complete removal of phospholipids. Such a size of the sample appears to be beneficial, considering the trace amount of xenobiotics or metabolites assayed in human plasma. 3.3. Comparison of efficiency of phospholipid removal cartridges In order to estimate the maximum amount of phospholipids that could be bonded on the tested cartridges, 1 mL of human plasma sample was applied on the SPE columns with different adsorbents: silica gel, Hybrid – phospholipid plates, Hybrid - PPT and silica modified with Cu(II). To quantify phospholipids, chromatographic conditions HPLC-DAD and LC-MS/MS were established and described in experimental part alongside with the procedure of sample preparation. The samples after SPE procedure were chromatographed with a mobile phase consisting of 10% methanol in water and monitored in the region between 190 nm and 400 nm. The results obtained are presented in Fig. 2. As it can be observed, the investigated samples possess a large matrix, which is eluted at 1–2 min. The fewest components were observed on chromatograms after the SPE procedures on silica modified with Cu(II), as compared to the remaining investigated cartridges. The purification achieved by new sorbent was further studied by LC-MS/MS method. Phospholipid fragment ions were used as markers for the effectiveness of sample preparation technique. There is a wide variety of glycerophosphocholines (GPCho’s) found in plasma (mouse, rat, rabbit, dog, monkey, and human). The lipid alkyl, acyl, and 1-alkenyl ester group 10

chain lengths may vary from 16–22 carbons and contain from 0 to 6 sites of unsaturation [21, 22]. Consequently, monitoring all ion transitions for these individual components in an MRM experiment is not practical. A large number of required transitions would decrease the method’s sensitivity. We have developed a method based on literature [18, 19] which is standard approach for testing the influence of phospholipids on the occurrence of matrix effects enables the detection of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) by monitoring MRM transitions common for all representatives of these classes (184→184 and 104→104 for PC and LPC, respectively). This MRM approach allows all GPCho’s to be monitored by just one transition in an MRM experiment. Our approach utilizes in-source collisionally induced dissociation (CID) to yield a common GPCho fragment ion (trimethylammonium-ethyl-phosphate ion; m/z 184). The resulting ion is then selected by quadrupole 1 (Q1), passed at low energy through collision cell (Q2) gas to avoid further fragmentation, and finally selected by quadrupole 3 (Q3). Since only voltage changes are employed, this MRM experiment may be utilized as part of a traditional MRM experiment to monitor drugs and metabolites within significant sensitivity loss. The content of phospholipids was converted into glycerophosphocholines via the m/z 184→184 transition and finally into 2-lyso-glycerophosphocholine, whose signal corresponds to 104 m/z (Fig. 3). Fig.4 (A) and (B) present percentage of these phospholipid fractions extracted from a human plasma sample by different techniques: protein precipitation method PPT, the HybridSPEPPT, HybridSPE-phospholipid, and SPE, applying silica gel modified with Cu(II). The effectiveness of plasma purification was the best for the last one. As it could be seen, the samples prepared using the silica modified with Cu(II) platform were depleted of 99% of the 2-lyso-glycerophosphocholine and almost 91% of the glycerophosphocholines from plasma sample when compared to standard protein precipitation (control). To evaluate the repeatability of phospholipids depletion by new extracting cartridges, plasma samples were

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analyzed in replicates (n=5), in short time intervals and under the same conditions (same equipment and operator). Results obtained were compared with LC-MS/MS measurements and good agreement was observed with RSD% 3.03 and 5.85 for both fractions of glycerophosphocholines and 2-lyso glycerophosphocholine respectively. The within-laboratory reproducibility was obtained by the same protocol, however, the analyses were performed on different days and by two different operators (n=5). Percentage of the relative standard deviation values for the measurements varied between 6.46 and 9.05 % for both respective phospholipid fractions. 3.4. FT-IR spectroscopy The FT-IR spectra of A) silica gel SPE cartridge, B) silica gel modified with Cu(II), C) silica gel modified with Cu(II) after the phospholipids retention onto the cartridge are presented in Fig. S4†. The characteristic bands for silica are present in sample A spectrum. The very broad bands at 3360 and 1632 cm-1 could be assigned to the stretching and bending vibrations of silanol groups, respectively [23]. The bands found at 1046 and 799 cm-1 are the characteristic antisymmetric and symmetric stretching modes of Si-O-Si, respectively. In the case of modified samples: Fig. S4†(B) and Fig. S4† (C), the later band is switched to the 788 cm-1, whereas, small and broad one was observed at 969 cm-1, which can be allocated to the Si-OH stretching mode, and in the case of modified samples this band appears as a shoulder [24]. 3.5. Raman spectroscopy and substrates distribution along z-axis The quality of Cu(II) deposition on silica was verified based on the comparison of the spectra: pure silica, silica modified with Cu(II) obtained from the confocal Raman Microscope. The results indicated a uniform distribution of Cu(II) on the surface and in the pores of adsorbent particles. In Fig. S5† the Raman spectra of investigate materials are presented. The visible

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differences between modified and unmodified columns are visible. For example, the bands around 425, 673, 3193 and 3292 cm-1 appear in the spectrum of silica gel modified with Cu(II) ions comparing with the pure silica gel (Fig. S5† (A) and (B) [25]. After isolation of phospholipids on the modified with Cu(II) silica gel cartridge the diminish of the band around 973 cm-1 and the appearance of the new band around 2982 cm-1 can be visible (Fig. S5† (C). In Fig. S6† the Raman depth images based on the Raman bands intensities obtained for silica gel related materials are presented. In the case of silica gel SPE cartridge, the uniform intensities of each bead due to the depth of measurement could be observed (Fig. S6† (A).The similar result was obtained for the silica gel modified with Cu(II) ions (Fig. S6† (B). The depth distribution of bands intensities varied most for the last material i.e. silica gel modified with Cu(II) ions and after phospholipid, isolation (Fig. S6† (C). What is interesting is the intensity of the bands increase along with the depth (the red color in the Fig. S6† (C), which may be evidence of phospholipid adsorption on the surface of modified silica gel beads and inside the porous structure of the beads. 4. Conclusion This manuscript examines a new phospholipid-removal approach that is simple and rapid. A new sorbent is based on silica gel modified with Cu(II) in the basic environment. The adsorbent proposed can be easily obtained in a process of percolation of copper (II) acetate in ammonia solution directly on silica particles. It is inexpensive and can be an alternative for commercially available cartridges for SPE. The implemented modification of silica gel was confirmed by the use of FT-IR and Raman spectroscopy. The Raman depth profiles allow accessing distribution of phospholipids inside mesoporous beads structure. Confocal Raman depth profiles showed that for silica gel with Cu(II), after the phospholipids isolation, the intensity of the bands increases with the depth, which can be evidence of phospholipids adsorption on the surface of modified silica gel beads and inside the porous structure of the 13

beads. New products have been investigated according to the specific capability of removing phospholipids. Elaborated SPE procedure utilizing silica gel modified with a copper (II) was compared to standard protein precipitation method and SPE applying for a phospholipidremoval plate and Hybrid SPE-PPT plates. The presence of phospholipids in eluates of each sample was monitored using m/z 184–184 and 104 m/z. Product designed was the most efficient at removing phospholipids from up to 1 mL of human plasma sample. Another advantage of silica gel modified with a copper (II) is expressed in the possibility of working in a basic pH range, which makes these cartridges suitable for bioanalytical assay of acidic compounds in single elution run.

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imine-modified silica for cyclohexane oxidation with hydrogen peroxide. RSC Adv., 4 (2014) 24820–24830. [25] S.H. Tohidi, A.J. Novinrooz, M. Derhambakhsh, G.L. Grigoryan, Dependence of Spectroscopic Properties of Copper Oxide Based Silica Supported Nanostructure on Temperature. Int. J. Nanosci. Nanotechnol., 8 (2012)143–148.

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Legend for figures: Fig. 1. Effect of the sample size on phospholipid removal by Cu (II) modified silica gel SPE cartridges and protein precipitation method (PPT). Peak area concerns the 2-lysoglycerophosphocholine content in the plasma sample. Fig. 2. 3D chromatograms of human plasma samples after SPE purification on I-Hybrid – phospholipid ultra, II-Hybrid –PPT, III-silica modified by Cu(II), obtained by HPLC using elution monitored at λ=190–400 nm. Fig. 3. Chromatograms of glycerophosphocholines (184 m/z) and 2-lysoglycerophosphocholines (104 m/z) of deproteinized serum: A) and after solid phase extraction on a variety of cartridges: B) Hybrid SPE Phospholipids Ultra C) Hybrid SPE-PPT D) Bakerbond SPE Silica Gel modified Cu. Fig. 4. (A) The percentage of the glycerophosphocholines (184 m/z, n=10) after using different plasma purification method. Error bars show standard errors of LC-MS/MS measurements. Fig. 4. (B) The percentage of the 2-lyso-glycerophosphocholine (104 m/z, n=10) after using different plasma purification method. Error bars show standard errors of LC-MS/MS measurements.

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Fig.1

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I

II

III

Fig.2

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A)

B)

C)

D)

Fig.3

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Fig.4 A

B

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