Microextraction of aromatic microbial metabolites by packed hypercrosslinked polystyrene from blood serum

Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112883

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Microextraction of aromatic microbial metabolites by packed hypercrosslinked polystyrene from blood serum Alisa K. Pautova a,b,∗ , Pavel D. Sobolev a , Alexander I. Revelsky a a

Laboratory of Mass Spectrometry, Chemistry Department, Lomonosov Moscow State University, GSP-1, Leninskie gory, 1-3, 119991, Moscow, Russia Laboratory of Human Metabolism in Critical States, Negovsky Research Institute of General Reanimatology, Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, Petrovka str., 25-2, 107031, Moscow, Russia b

a r t i c l e

i n f o

Article history: Received 13 August 2019 Received in revised form 10 September 2019 Accepted 13 September 2019 Available online 14 September 2019 Keywords: Phenylcarboxylic acids Solid-phase extraction Microextraction by packed sorbent Hypercrosslinked polystyrene Gas chromatography-mass spectrometry Aromatic microbial metabolites

a b s t r a c t The article is devoted to the application of modern sample preparation technique - microextraction by packed sorbent (MEPS) - in conjunction with non-conventional type of sorbent - hypercrosslinked polystyrene, that was investigated for the first time in this work. Their combination was used to extract phenylcarboxylic acid-type aromatic microbial metabolites from serum samples of a healthy volunteer with following derivatization and GC–MS detection. As barrel insert and needle for MEPS with hypercrosslinked polystyrene is not produced, we designed a device to imitate the commercial MEPS system with packed granular biporous hypercrosslinked polystyrene. Nine aromatic microbial metabolites, including sepsis associated phenyllactic, 4-hydroxyphenyllactic and 4-hydroxyphenylacetic acids, were extracted from serum samples (recoveries were 20–70%) and a linear dependence was revealed in the most clinically significant range of concentrations (0.5–18 ␮M). The results obtained demonstrate the perspective of the applying of hypercrosslinked polystyrene in commercial devices for MEPS for the future analyses of biological samples, in particular for the early diagnosis of sepsis and treatment effectiveness control. © 2019 Elsevier B.V. All rights reserved.

1. Introduction New methods of sample preparation techniques are important tools for the development of metabolomics approaches based on mass spectrometry [1]. Microextraction by packed sorbent (MEPS) is a promising alternative for solid-phase extraction (SPE). Its main advantages are reduced amount of solvent and sample volumes, and repeated use of the packed syringe for more than 100 times. Common for SPE sorbent materials are commercially available for MEPS. They are silica based (C2, C8, C18), restricted access materials, polystyrene divinylbenzene polymer et al. [2–5].

Abbreviations: BA, benzoic acid; BIN, barrel insert and needle; GC–MS, gas chromatography-mass spectrometry; HCLPS, hypercrosslinked polystyrene; HVA, homovanillic acid; MEPS, microextraction by packed sorbent; p-HBA, 4hydroxybenzoic acid; PhCA, phenylcarboxylic acid; PhPA, phenylpropionic acid; PhLA, phenyllactic acid; p-HPhAA, 4-hydroxyphenylacetic acid; p-HPhLA, 4hydroxyphenyllactic acid; p-HPhPA, 3-(4-hydroxyphenyl)propionic acid; RSD, relative standard deviation; SPE, solid-phase extraction; TIC, total ion current; TMS, trimethylsilyl. ∗ Corresponding author at: Petrovka str., 25-2, Moscow, 107031, Russia. E-mail address: [email protected] (A.K. Pautova). https://doi.org/10.1016/j.jpba.2019.112883 0731-7085/© 2019 Elsevier B.V. All rights reserved.

In our previous studies we have demonstrated that MEPS with C18 can be an effective method for the extraction of some aromatic microbial metabolites (AMM) from model solutions [6] and serum samples of healthy people and critically ill patients [7]. These AMM of phenolic structure (phenylcarboxylic acids – PhCAs) are metabolites of tyrosine and phenylalanine and their concentration in serum of septic patients can change for tens of times in comparison with their concentrations in serum of healthy people. The concentrations of three sepsis-associated PhCAs correlate with the severity of condition and symptoms of sepsis: phenyllactic acid (PhLA), 4hydroxyphenyllactic acid (p-HPhLA), and 4-hydroxyphenylacetic acid (p-HPhAA) [8,9]. Based on these data, new techniques were developed to control treatment effectiveness in critically ill patients [10,11]. Some PhCAs (benzoic (BA), phenylpropionic (PhPA), PhLA and p-HPhLA) are constantly present at low concentrations in blood serum of healthy people [9,12], which reflects the integration of human metabolism and its microbiota [8]. Initially liquid-liquid extraction was used as sample preparation technique [12,13], but MEPS showed more performance simultaneously with comparable recoveries and reproducibility [7]. Hypercrosslinked polystyrene (HCLPS) developed by prof. Davankov in 1970s arouses much interest for sample preparation of various complex matrixes, including urine and blood. HCLPS

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can be utilized for the preferential adsorption of small molecules rather than largely, rejected proteins and other spacious molecules [14–16]. We have investigated and demonstrated that SPE with HCLPS irregular sorbent particles with 50–100 ␮m size fraction and 200% crosslinking degrees (Pyrosep 270, Purolite International, UK) can be used for the extraction of AMM from model solutions [17]. Information on the application of HCLPS for MEPS was not found. The goal of the study presented herein was to develop the conditions of microextraction of the PhCAs from model solutions and serum samples using packed HCLPS followed by derivatization and GC–MS detection. 2. Materials and methods 2.1. Samples and materials Benzoic acid (BA, ≥99.5%), 2,3,4,5,6-D5 -benzoic acid (D5 BA, ≥99 atom % D, ≥99%), phenylacetic acid (PhAA, ≥98%), cinnamic acid (≥98%), phenylpropionic acid (PhPA, ≥99%), phenyllactic acid (PhLA, ≥98%), 4-hydroxybenzoic acid (pHBA, ≥99%), 4-hydroxyphenylacetic acid (p-HPhAA, ≥98%), 4-hydroxyphenylpropionic acid (p-HPhPA, ≥98%), homovanillic acid (HVA, ≥97%), 4-hydroxyphenyllactic acid (p-HPhLA, ≥97%), N,O-bis(trimethylsilyl)trifluoroacetamide (99%, contains 1% trimethylchlorosilane), formic acid (≥95%), methyl-tert-butyl ether (≥99.8%), hexane (≥97.0%), methanol (≥99.9%) were obtained from Merck (Germany); sulfuric acid, acetone, diethyl ether, sodium chloride were Laboratory Reagent grade and obtained from Khimreactiv (Russia). Granular biporous HCLPS was synthesized by our colleagues from Nesmeyanov-Institute of Organo-Element Compounds, Russian Academy of Sciences. It was obtained by crosslinking macroporous styrene copolymer (93%) with divinylbenzene (7%) (crosslinking degree 200%). Apparent specific inner surface area ∼1100 m2 /g; overall pore volume ∼ 1.2 ml/g, micropore volume ∼ 0.4 ml/g, mesopore volume ∼ 0.8 ml/g; average micropore size ∼ 15 Å, average macro- and mesopore size ∼ 880 Å; granule size – 40–80 ␮m. Serum samples from healthy volunteers were collected in Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology (Moscow, Russia), local ethical committee approval was received. Serum samples were frozen and stored at −30 ◦ C for not more than one month. Serum samples were defrosted at room temperature prior to use.

2.2. Preparation of model solutions Preparation of model solutions of the PhCAs and internal standard was described in a previous study [7]. Briefly, we used the mixture of all PhCAs in acetone (6 mg/l) and acetone solution of the internal standard (7.5 mg/l) to perform the experiments without any kind of sample preparation except derivatization. For derivatization, a 5 ␮l aliquot of acetone solution of the PhCAs and a 5 ␮l aliquot of acetone solution of internal standard were dissolved in 70 ␮l of diethyl ether. The concentration of each PhCA in this solution was equal to 375 ␮g/l (2–3 ␮mol/l), the internal standard concentration – 469 ␮g/l. The solution was evaporated at 40 ◦ C. A 20 ␮l aliquot of N,O-bis(trimethylsilyl)trifluoroacetamide was added to the precipitate to form trimethylsilyl (TMS) derivatives (15 min, 80 ◦ C). The mixture was cooled at +4 ◦ C, dissolved with 60 ␮l of hexane and 1 ␮l of the solution was analyzed by GC–MS. To investigate PhCA extraction from model solutions and serum samples, aqueous stock solutions of each PhCA (6 mg/l) and internal standard (7.5 mg/l) were prepared. Aliquots (5 ␮l) of aqueous solutions of the PhCAs (6 mg/l) and standard (7.5 mg/l), concentrated sulfuric acid (2.5 ␮l) and distilled water (70 ␮l) were added to 80 ␮l of water during the experiments with model solutions or to 80 ␮l of serum sample of a healthy volunteer. The stock solutions of the PhCAs were then used to prepare solutions with concentrations of 94, 375, 750 and 2250 ␮g/l to create respective calibration curves. Some PhCAs are ubiquitous in the serum of healthy individuals, that was taken into account during the experiments. The least squares method was used to calculate the equations of the calibration curves. 2.3. Microextraction by packed HCLPS A device for microextraction by packed HCLPS was designed. A piece of quartz wool was placed and compressed at the tip of the Pasteur glass pipette (1.5 ml). HCLPS (5 g) was mixed with acetone (5 ml) and 25 ␮l of the mixture (2 mg of sorbent) were put into the Pasteur glass pipette. Then the pipette was washed thoroughly with acetone. Another piece of compressed quartz wool fixed the sorbent. The pipette was attached to a medical syringe (1 ml) with a small piece of rubber tubing (about 2 cm). The parameters of the microextraction by packed HCLPS that were investigated here included: pH of model solution, number of sorption/desorption cycles and the eluent volume. Final optimal conditions for sample preparation from model solution and serum samples are: • Model solution: water (70 ␮l), internal standard (5 ␮l, 7.5 mg/l), PhCA solution (5 ␮l, 6 mg/l), concentrated sulfuric acid (2.5 ␮l); • Serum sample preparation: water (70 ␮l), blood serum (80 ␮l), internal standard (5 ␮l, 7.5 mg/l), PhCA solution (5 ␮l, 6 mg/l), concentrated sulfuric acid (2.5 ␮l); • HCLPS conditioning: acetone, water (2 × 80 ␮l); • Sample loading: model solution or serum sample solution (2 × 80 ␮l); • Sorbent washing: 0.3 mmol/l formic acid solution (1 × 80 ␮l); • Drying: passing the air though the sorbent (3 × 80 ␮l); • Elution: diethyl ether (5 × 40 ␮L); • Evaporation; • Derivatization; • GC–MS analysis. 2.4. GC–MS analysis

Fig. 1. Developed device for microextraction by packed HCLPS.

GC–MS analyses were performed on a Trace GC 1310 gas chromatograph equipped with ISQ LT mass spectrometer obtained from Thermo Scientific (Thermo Electron Corporation, USA). The

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Fig. 2. Chromatograms (TIC) of serum samples of a healthy volunteer after SPE (a), liquid-liquid extraction (c) and mass chromatogram of serum sample of a healthy volunteer after SPE (b) obtained using m/z values of the PhCA TMS derivatives (104, 147, 164, 179, 192, 193, 207, 296).

first part of our study on SPE using HCLPS was performed using two capillary columns: Restek RTX1 ms quartz capillary column (100% dimethylpolysiloxane, thickness of steady phase 0.25 ␮m, length of 30 m, internal diameter of 0.32 mm) and HP 1 quartz capillary column (100% dimethylpolysiloxane, thickness of steady

phase 0.11 ␮m, length of 7 m, internal diameter of 0.2 mm). The second part of our study on microextraction by packed HCLPS with expanded number of analytes (including BA, cinnamic acid and polar HVA and p-HBA) was performed using the capillary column TR-5 ms (95% poly(dimethylsiloxane) + 5% phenyl

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Table 1 Statistical data for F- and Student’s t-tests of the equality of two series of experiments for two devices for microextraction by packed HCLPS (model solutions, concentrations of the PhCAs – 375 ␮g/l, P = 0.95, n = 3). Parameter Device #1

Device #2

Mean recovery Variance Mean recovery Variance

F-test t-test

BA

PhPA

Cinna-mic

PhLA

p-HBA

p-HPhAA

p-HPhPA

HVA

p-HPhLA

53

64

29

21

19

23

24

25

8

13 53 2 6.21 0.02

36 58 50 1.40 1.11

34 27 12 2.98 0.50

22 24 33 1.46 0.58

15 20 18 1.16 0.36

26 27 24 1.08 1.03

39 26 25 1.57 0.45

19 27 15 1.28 0.64

4 7 4 1.15 0.59

polysilphenylene-siloxane phase, 30 m ×0.25 mm, df =0.25 ␮m). Constant gas flow was 1.5 ml/min; the carrier gas was helium, split 1:20. GC analysis was performed in 25 min with a starting oven temperature 80 ◦ C (hold time 4 min) and a single ramp of 10 ◦ C/min to 250 ◦ C (hold time 4 min). The injector temperature was 200 ◦ C and injection volume was 1 ␮l. Full-scan mass spectra were recorded with m/z range 50–480 in electron ionization mode at 70 eV, using Xcalibur 2.2 software. The MS source was 200 ◦ C and the GC–MS interface was kept at 250 ◦ C. Scan rate was 3 scans/sec; cathode delay time 4 min. Retention times and characteristic m/z values of TMS derivatives of the PhCAs have been described in detail in our previous papers [7,12]. Mass spectra data for the TMS derivatives of the PhCAs were proved by the NIST mass spectra library. The PhCA recoveries were calculated using formula 1: Recovery(%) = (APhCAs /ASt )/(APhCAs /ASt )100% ×100,

(1)

APhCAs – peak areas of the PhCAs; ASt – D5 -BA area (internal standard); (APhCAs /ASt ) – relative signal obtained after sample preparation; (APhCAs /ASt )100% – relative signal obtained without any kind of sample preparation (except derivatization). Statistical processing of data was performed based on the results of three parallel experiments using Microsoft Excel 2013. All data represented the mean recoveries with a confidence interval (P = 0.95, n = 3). Relative standard deviation (RSD, %) was used to compare reproducibility of the results. 3. Results and discussion The first part of our study on SPE using HCLPS logically continued our previous study on the development of the conditions of the PhCA extraction from model solutions [17]. HCLPS Pyrosep 270 irregular sorbent particles with size distribution of 30–60 ␮m were applied (the same properties as MN 270). The main steps of the sample preparation were the following: • Volume of the sorbent: 300 ␮l; • pH of model solution: 7; • Sorbent conditioning: acetone, water, 1 × 10−2 mol/l sulfuric acid solution; • Washing solution: 1 × 10− 5 mol/l sulfuric acid solution (3 ml); • Drying of the sorbent; • Elution: methyl-tert-butyl ether (2 × 600 ␮l). In order to examine the efficiency of the developed SPE conditions from serum samples of a healthy volunteer, 200 ␮l of serum were diluted with 800 ␮l of distilled water, solid sodium chloride was added for the protein denaturation. Subsequently, the sample was centrifuged for 5 min at 6000 rpm, the supernatant was decanted and subjected to the developed sample preparation conditions. The chromatogram in total ion current (TIC) is presented in Fig. 2a. The peaks of TMS derivatives of five PhCAs (PhPA, PhLA, p-HPhAA, p-HPhPA, p-HPhLA) are identified on the

mass chromatogram (Fig. 2b) mostly at the level of detection limit. When comparing the chromatogram (TIC) obtained after the SPE of the serum sample of a healthy volunteer with the chromatogram (TIC) obtained previously in our laboratory after the liquid-liquid extraction [12] of the serum sample of the same healthy volunteer (Fig. 2c), we can judge on the higher effectiveness of the SPE with HCLPS for the separation of target PhCAs from interfering components of blood serum, the cholesterol in particular (tr = 28.18 min on Fig. 2c): the peak of cholesterol on the chromatogram after the liquid-liquid extraction (tr = 14.97 min on Fig. 2a) is noticeably higher (cholesterol retention times varied due to analyses on capillary columns of different lengths). The interfering matrix components as cholesterol contaminated the GC–MS system. This at least required additional column oven heating at the maximum acceptable by column temperature, additional reference tests with extra reference tests with the addition of pure solvents and high inlet liner replacement frequency. This result confirmed our previous results on model solutions where we observed the recovery of the PhCAs more than 50% together with the recovery of the cholesterol less than 10%. At the same time, most of the cholesterol was found in the washing solution [17]. Despite these very promising results the recoveries of the PhCAs were irreproducible. We suppose that this could be related to the contamination of the HCLPS after the SPE from serum samples. Moreover, it was impossible to carry out more than 1 analysis per one cartridge per day. Water, acetone and sulfuric acid solution (pH 2) were initially used as washing solvents during the regeneration step. Different variations of the sorbent regeneration were investigated unsuccessfully, however, the complete regeneration of the sorbent was achieved only after it was kept in acetone overnight. The decision was found in usage of lesser amounts of another modification of HCLPS sorbent for MEPS – granular biporous HCLPS with 200% crosslinking degrees, that was synthesized by our colleagues from Nesmeyanov-Institute of Organo-Element Compounds, Russian Academy of Sciences. MEPS is one of the most promising modern variations of SPE that uses small amount of sorbent. Unfortunately, as far as we know, MEPS barrel insert and needle (BIN) with HCLPS is not produced. We designed the device for MEPS with HCLPS (Fig. 1). When the syringe plunger moves up, a sample passes through the sorbent and target components are sorbed; when the plunger moves down, the sample solvent leaves the syringe. The sample can pass through the sorbent several times. Thus, this device is an imitation of commercial MEPS system. Moreover, the list of the AMM under investigation was expanded and other potentially clinically significant acids like HVA, p-HBA, BA and cinnamic acid were added to the further experiments [12]. The conditions for microextraction by packed HCLPS were selected based on the conditions of described SPE technique and obtained in our previous study on the PhCA extraction from serum samples with MEPS using C18 [7]: • Sample dilution: 80 ␮l of serum sample, 75 ␮l of water, 5 ␮l of internal standard, pH 2;

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Fig. 3. Recoveries of the PhCAs from model solutions with pH 2 and pH 7 after microextraction by packed HCLPS (concentration of each acid – 375 ␮g/l, P = 0.95, n = 3). Table 2 Recoveries (%) of the PhCAs from serum samples of a healthy volunteer using microextraction by packed HCLPS (concentrations of the PhCAs – 375 ␮g/l) and the calibration curve equations in clinically significant range of concentrations (94–2250 ␮g/l; 0.5–18 ␮mol/l). PhCA BA PhPA Cinnamic PhLA p-HBA p-HPhAA p-HPhPA HVA p-HPhLA

Recovery, % 45 ± 20 50 ± 20 35 ± 20 70 ± 15 45 ± 20 45 ± 10 40 ± 20 40 ± 10 20 ± 10

The equations of calibration curves −3

y = (3.8 ± 0.3) × 10 x + (0.2 ± 0.3) y = (2.7 ± 0,1) × 10−3 x − (0.1 ± 0.1) y = (1.3 ± 0.1) × 10−3 x − (0.2 ± 0.1) y = (1.4 ± 0,1) × 10−3 x − (0.1 ± 0.1) y = (2.7 ± 0.1) × 10−3 x − (0.3 ± 0.2) y = (1.5 ± 0.1) × 10−3 x − (0.2 ± 0.2) y = (3.9 ± 0.3) × 10−3 x − (0.6 ± 0.4) y = (1.6 ± 0.1) × 10−3 x − (0.2 ± 0.2) y = (2.0 ± 0.1) × 10−3 x − (0.2 ± 0.1)

• Cartridge conditioning: methanol, water, 3 mmol/l formic acid solution (3 × 50 ␮l, 900 ␮l/s); • Sample loading: 15 × 50 ␮l, 300 ␮l/s; • Sorbent washing: 0.3 mmol/l formic acid solution (2 × 20 ␮l, 500 ␮l/s); • Drying: air (5 × 50 ␮l, 900 ␮l/s); • Elution: diethyl ether (10 × 10 ␮l, 600 ␮l/s). Based on these data the initial conditions for microextraction of the PhCAs from model solutions by packed HCLPS were the following: • HCLPS conditioning: acetone, water (2 × 80 ␮l); • Sample loading: model solution of the PhCAs (pH neutral, 5 × 80 ␮l); • Sorbent washing: 0.3 mmol/l formic acid solution (1 × 80 ␮l); • Drying: passing the air though the sorbent (3 × 80 ␮l); • Elution: diethyl ether (3 × 80 ␮l).

R2 0.9967 0.9998 0.9956 0.9978 0.9977 0.9935 0.9947 0.9946 0.9993

While two devices were assembled, we decided to compare them using model solutions. Three replicates were performed using each syringe. Results were compared statistically using F-test and Student’s t-test (Table 1). The F calculated from the data for all PhCAs were not greater than critical value of the F-distribution (F(0.95; 2; 2) = 19) and, hence, two variances are considered equal. The t calculated from the data for all PhCAs also were not greater than critical value of the t-distribution (t(0.95; 4) = 2.78) and, hence, the two means are considered equal. Thus, the results obtained using two devices for the microextraction by packed HCLPS are comparable. The pH value of model solution was previously shown to be an important parameter which influences the PhCA sorption. The experiments with pH 2 and pH 7 (Fig. 3) demonstrated the absence of statistically significant differences in the recoveries of the PhCAs (except p-HPhLA). The recoveries of the PhCAs at pH 2 were 20–60%, at pH 7 – 10–60%. However, reproducibility of the results was much better for pH 2 (RSD = 5–15%) than for pH 7 (RSD = 5–25%).

Fig. 4. Recoveries of the PhCAs from serum samples of healthy volunteer using MEPS with C18 and microextraction by packed HCLPS (concentration of each acid – 375 ␮g L−1 , P = 0.95, n = 3).

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Fig. 5. Mass chromatogram of a serum sample from a healthy volunteer with the addition of the PhCAs (concentration of each acid – 375 ␮g/l) after microextraction by packed HCLPS obtained using m/z values of the TMS derivatives (104, 105, 179, 192, 193, 223, 267, 296, 326).

As p-HPhLA is one of the most clinically significant PhCAs, further experiments with serum samples were performed after acidification to pH 2 and dilution in 2 times as it was recommended for MEPS [3] and used in our experiments with C18 [7]. The following parameters of sorption/desorption cycles investigated: 2 × 80 ␮l/3 × 80 ␮l; 5 × 80 ␮l/1 × 80 ␮l; were 5 × 80 ␮l/3 × 80 ␮l; 5 × 80 ␮l/5 × 40 ␮l. The highest PhCA recoveries (for more non-polar acids – BA, PhPA, cinnamic acid – 30–50%, the other PhCAs – 40–70%) were obtained applying the last conditions of sorption/desorption (5 × 80 ␮l/5 × 40 ␮l). The other conditions led to the recoveries of more polar acids 20–60 %. Particularly the results for 2 × 80 ␮l/3 × 80 ␮l and 5 × 80 ␮l/3 × 80 ␮l were comparable. To reduce the time of sample preparation we chose 2 × 80 ␮l for sorption and 5 × 40 ␮l for desorption for our further experiments with the serum samples of healthy volunteer. The PhCA recoveries from serum samples using microextraction by packed HCLPS are presented in Table 2. The comparison of the PhCA recoveries from serum samples after MEPS with C18 and microextraction by packed HCLPS is demonstrated in Fig. 4. The recovery of p-HPhLA was higher using MEPS with C18; the recoveries of other acids were similar for both techniques. All PhCAs are clearly identified on the mass chromatogram (Fig. 5). The reproducibility of the results for commercial MEPS system with C18 was better (RSD = 5–10%) than for our device with packed HCLPS (RSD = 10–20%). It should be noted that the reproducibility for HCLPS could be significantly improved by using commercial MEPS systems with standardized BINs. Calibration curves were calculated using serum samples of a healthy volunteer in the 94–2250 ␮g/l (0.5–18 ␮mol/l) concentration range, where ≤ 94 ␮g/l (≤0.5–0.8 ␮mol/l) is the AMM content in healthy person’s serum [12] and 375–2250 ␮g/l (2–18 ␮mol/l) is the most clinically significant concentration range of the AMM, which allows to make conclusions about the presence of an infectious complication and the severity of a patient’s condition. The calibration equations for each of the PhCAs are linear functions in the considered concentration range (R2 ≥0.9935) (Table 2), which makes possible the utilization of HCLPS for MEPS of the analytes in serum samples in case of presence of commercial MEPS BINs. 4. Conclusion We investigated different types of HCLPS for the extraction of AMM from serum samples. The matrix effect from less volatile serum components (for instance, cholesterol), which are usu-

ally co-extracted with PhCAs during liquid-liquid extraction, was largely reduced. Limitations of SPE from serum samples with HCLPS Pyrosep 270 irregular sorbent particles were demonstrated, and the advantages of the granular biporous HCLPS for the MEPS were revealed. All PhCAs, including sepsis-associated PhLA, p-HPhAA and p-HPhLA, were successfully extracted from serum samples in clinically significant range of concentrations using microextraction by packed HCLPS. Application of HCLPS in commercial MEPS devices will probably improve the reproducibility of the obtained results. This hypothesis is indirectly confirmed by the recently published article of Davankov et al. where new modification of HCLPS was used for SPE [18]. The capabilities of this new sorbent for sorption in static mode were presented in another research [19]. Furthermore, HCLPS is known to be used as hemosorbent for the blood and plasma detoxification in the treatment of sepsis [20]. This fact is interesting and important as some PhCAs could be not just microbial metabolites but also toxins as they influence the cellular respiration, the function of mitochondrial enzymes, the phagocytosis activity, the ability of leucocytes to produce active oxygen forms, etc. [21,22], that is the evidence of a direct role of the PhCAs in the pathogenetic mechanisms of critical conditions. According to these data we assume that the results on the sorption/desorption of the PhCAs on HCLPS are important for the development of the new approach to their automated extraction from serum samples for the sepsis diagnosis and for understanding of the blood detoxification mechanisms using HCLPS. Declaration of Competing Interest None. Acknowledgments This work was supported by Russian Science Foundation [grant number 15-15-00110-P]. The authors are grateful to MS Analitika Company for providing the GC–MS system. References [1] N.L. Kuehnbaum, P. Britz-McKibbin, New advances in separation science for metabolomics: resolving chemical diversity in a post-genomic era, Chem. Rev. 113 (2013) 2437.

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