Authors:
Tomasz Tuzimski Department of Physical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093, Lublin, Poland

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Michał Szubartowski Student Research Group at the Department of Physical Chemistry, Chair of Chemistry, Faculty of Pharmacy, Medical University of Lublin, Chodźki 4a, 20-093, Lublin, Poland

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Abstract

In this study, we propose a simple, cost-effective, and sensitive high-performance liquid chromatography with both detection techniques such as diode-array detection and fluorescence detection (HPLC-DAD-FLD) for the determination of nesfatin-1 in fetal bovine serum samples. The limit of detection (LOD) and limit of quantification (LOQ) for nesfatin-1 were set at satisfactory values in the range 0.22–0.35 mg mL−1 and in the range 0.67–1.05 mg mL−1, respectively (at two different wavelengths (DAD) and at four different wavelengths (FLD)). Analyte concentrations were determined as the average value from fetal bovine serum matrix samples. The preliminary results show that the SPE procedure on Isolute Si-TsOH (SCX-3) could be used for further nesfatin-1 analyses in human serum samples. Both the SPE technique, chromatographic analysis with gradient elution mode and detection technique are fast and convenient.

Abstract

In this study, we propose a simple, cost-effective, and sensitive high-performance liquid chromatography with both detection techniques such as diode-array detection and fluorescence detection (HPLC-DAD-FLD) for the determination of nesfatin-1 in fetal bovine serum samples. The limit of detection (LOD) and limit of quantification (LOQ) for nesfatin-1 were set at satisfactory values in the range 0.22–0.35 mg mL−1 and in the range 0.67–1.05 mg mL−1, respectively (at two different wavelengths (DAD) and at four different wavelengths (FLD)). Analyte concentrations were determined as the average value from fetal bovine serum matrix samples. The preliminary results show that the SPE procedure on Isolute Si-TsOH (SCX-3) could be used for further nesfatin-1 analyses in human serum samples. Both the SPE technique, chromatographic analysis with gradient elution mode and detection technique are fast and convenient.

Introduction

Nesfatin-1 is an 82 amino acids long peptide derived from a parent protein, nucleobindin-2 (NUCB2), as a result of proteolysis by prohormone convertase enzymes, PC1/3 and PC2 at conserved cleavage sites, Lys-Arg and Arg-Arg, respectively [1]. Nesfatin-1 (Nucleobindin2-encoded satiety- and fat-influencing protein 1) was first identified in rat as a hypothalamic satiety-inducing peptide [1] and thereafter its presence has been demonstrated in several tissues of mammalian and non-mammalian vertebrates except for reptiles [2]. It has been shown to regulate food in-take, gastrointestinal activity, glucose homeostasis, cardiovascular functions, adipocyte growth and differentiation, reproduction and thermoregulation [3]. In addition, nesfatin-1 [4] has been linked to various diseases such as obesity, depression, anxiety, and epilepsy.

Nucleobindin 2 (NUCB2) was first described in 1992 [5]. Nucleobindin-2 (NUCB2) is a precursor protein of nesfatin-1 (NESF-1). Since NUCB2 and NESF-1 are colocalized in each tissue, their expression is often analysed together as NUCB2 [6]. The metabolic function of NUCB2/NESF-1 is related to food intake, glucose metabolism, and the immunoregulation/the immune, cardiovascular and endocrine systems [6–10]. The NUCB2/NESF-1 gene in humans is located on chromosome 11 and consists of 14 exons spanning 54,785 nucleotides [6, 11]. Nucleobindin-2 is a 396-amino acid protein preceded by a 24-amino acid signal peptide [6, 12]. The protein is proteolytically cleaved by the prohormone convertase. As a result, N-terminal nesfatin-1, nesfatin-2, and the C–terminal nesfatin-3 are formed [1, 8]. The functions of nesfatin-2 and the C–terminal nesfatin-3 have not been understood yet [6, 13].

Recently, it has been demonstrated that high expression of NUCB2/NESF-1 is associated with poor outcomes and promotes cell proliferation, migration, and invasion in, e.g., breast, colon, prostate, endometrial, thyroid, bladder cancers, or glioblastoma [6]. Interestingly, nesfatin-1 is also considered an inhibitor of the proliferation of human adrenocortical carcinoma and ovarian epithelial carcinoma cells. These conflicting results make NUCB2/NESF-1 an interesting target of study in the context of cancer progression [6]. Cancer is a heterogeneous disease, and even tumors with similar clinicopathological characteristics show different biology, behavior, and treatment responses [14].

Despite the increasing knowledge about the expression and regulation of NUCB2/NESF-1, its role in physiology and pathology is still poorly understood. Nucleobindin-2/Nesfatin-1 is also a component of various secreted body fluids, including saliva, synovial fluid, milk, and plasma or serum [15, 16]. Therefore, there is a nowadays need to define new prognostic and predictive markers to make treatment options more personalized [6, 17].

In the present work, a low cost and fast method for identification and quantitative analysis of nesfatin-1 in fetal bovine serum samples is proposed. After further optimization, the procedure might be dedicated to the routine biomonitoring of nesfatin-1 in human serum samples.

Experimental

Standard of the Nesfatin-1

Standard of the nesfatin-1 (Nesfatin-1 Human E. coli, recombinant protein, ref. RD172227500) was purchased from BioVendor – Laboratorni medicina a.s. (Brno, Czech Republic). Individual stock nesfatin-1 standard solutions (0.1, 0.25, 0.5, 0.75, 1 mg mL−1) were prepared in 0.1% trifluoroacetic (TFA) or 0.1% trichloroacetic (TCA) water solution and were stored at −20 °C to −80 °C. A nesfatin-1 standard solutions were analysed directly as the same day by HPLC–DAD-FLD.

Solvents and mobile-phase solutions

TFA suitable for HPLC ≥99.0% (CAS: 76-05-1) and TCA, ACS reagent, ≥ 99.0% (CAS: 76-03-9) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

LC-MS grade methanol (MeOH), the gradient grade for liquid chromatography acetonitrile (MeCN) and formic acid were obtained from E. Merck (Darmstadt, Germany); LC-MS grade water was purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water (0.07–0.09 mS cm−1) was produced in our laboratory using a Hydrolab System (Gdańsk, Poland).

Apparatus and HPLC-DAD-FLD conditions

An Agilent Technologies 1200 HPLC system with a quaternary pump and an autosampler with thermostat samples was used for the LC analysis. Analytes were separated using a PL Rapide Aqua L 150 × 7.5 mm column with a 3-µm particle size (Agilent Technologies, Stockport, UK). The column was thermostated at 22 °C. The mobile phase consisted of 0.1% TCA in water and 0.1% TFA in water (70 : 30, v/v, component A) and consisted of 0.1% TCA in acetonitrile and 0.1% TFA in acetonitrile (64 : 36, v/v, component B) in a gradient elution: 0–30 min from 5% eluent B to 100% B; 30–45 min isocratic 100% B.

The mobile phase flow rate was 1 mL min−1. In order to elute interferences of the matrix, before the next step of fetal bovine serum samples analysis, the isocratic elution with 100% B as the mobile phase was applied for 15 min (30–45 min) with a flow rate of 1 mL min−1, and the next isocratic elution had the initial conditions.

DAD and FLD conditions

Detection was carried out simultaneously at five different wavelengths such as 220, 230, 240, 275 and 280 nm and with the following detector settings: bandwidth 4; reference wavelength 380; reference bandwidth 100.

FLD detection was carried out simultaneously at four different excitation wavelengths 200, 230, 240 and 250 nm. The emission wavelength was set at 300 nm during all studies. Other FLD detector settings were as follows: peakwidth >0.2 min (4 s resp. time; 2.31 Hz); multiple wavelengths: multi excitation: 230, 240, 250 nm; acquire spectra: all in the peak (step 10 nm); detection mode: fluorescence mode; fluorescence scan range: excitation from 200 to 300 nm and step 10 nm; emission from 310 to 340 nm and step 10 nm.

Analyse of the Nesfatin-1 by HPLC-DAD-FLD

Identification of nesfatin-1 was accomplished on the basis of the retention time of the analyte and by comparison between the UV spectra of the reference compounds in the chromatograph library and the UV spectra of the detected peaks in the samples.

Quantitative analysis in this regard was carried out on the basis of single-point calibration, in which response factors were calculated as amount-to-area ratios of the analytes in the calibration sample and used in the analyte-concentration calculation in spiked samples.

A validation study was performed using spiked fetal bovine serum samples and included evaluation of the selectivity, linearity, limits of detection (LODs), limits of quantification (LOQs).

Selectivity

The selectivity was evaluated by analyzing the fetal bovine serum samples from different sources to investigate potential interferences with the signals of analytes. The extent of interferences originating from endogenous fetal bovine serum sample components at the specific retention time of each analyte was evaluated through a comparison of an average blank fetal bovine serum samples matrix sample with the spiked fetal bovine serum (blank) matrix samples. HPLC analyses of nesfatin-1 standards were repeated three times. The identification of nesfatin-1 was accomplished on the basis of the retention time of the analyte.

Linearity

The linearity of the method was studied by spiking the fetal bovine serum samples with suitable amount of nesfetin-1 standard solution. Samples were prepared according to SPE and determined by the HPLC-DAD-FLD method described in the Experiment section.

Calibration curves for the LOD and LOQ values were constructed by analyzing nesftain-1 standard in 0.1% TFA in H2O at five concentrations, from 0.1 to 1 mg mL−1, using two replicates. The calibration curves were obtained by means of the least-squares method.

The limits of detection (LODs) and limits of quantification (LOQs) obtained for nesfatin-1 were calculated according to the formulas LOD = 3.3 (SD/S), and LOQ = 10 (SD/S), where SD is the standard deviation of the response (peak area) and S is the slope of the calibration curve. HPLC-DAD-FLD analyses of nesfatin-1 standards were repeated three times.

Sample preparation

Preparation of fetal bovine serum samples before SPE

The conditions for the extraction of nesfatin-1 from fetal bovine serum samples were optimized using the best, most optimal variant of sample preparation, i.e., to 100 µL of the fetal bovine serum sample was added 100 µL of a mixture of standard nesfatin-1 of 1 mg mL−1 concentration diluted in 1% TFA in water.

SPE-based extraction procedure

SPE was performed using Isolute SCX-3 cartridges containing 50 mg sorbent and a Baker SPE-12G SPE chamber (J.T. Baker, Phillipsburg, NJ). Before use, each cartridge was conditioned with 300 µL 1% TFA/H2O. Next, 100 µL fetal bovine serum sample diluted in solution of 1% TFA in water (v/v) was loaded onto the SPE column (negative pressure, 750 mbar on the pump). Afterward, the SPE columns were dried for 1 min (left under vacuum pressure in the SPE chamber), and then eluted to 200 µL of elution mixture (25% ammonia + acetonitrile + water, 20 : 20 : 60, v/v/v) in a 0.1 mL micro-insert (AnchemPlus, clear glass sillanized, Warsaw, Poland). Extracts were analysed directly as the same day by HPLC–DAD-FLD.

Calculation of relative standard deviation values (RSD) and extraction recovery

Accuracy in all cases was expressed as percentage recovery of the analyte using equation:
Recovery% = Average analyte concentration found in the spiked fetal bovine serum sample Analyte concentration added to the spiked fetal bovine serum sample × 100 %
Precision was expressed as RSD% calculated as follows:
RSD% = Standard deviation of the recovery  ( % ) Mean recovery  ( % ) × 100 %

Results and discussion

The application of HPLC-DAD-FLD after SPE for identification and quantitative analysis of nesfatin-1 in fetal bovine serum samples is demonstrated. Nesfatin-1 was determined with application of chromatographic system containing as stationary phase the PL Rapide Aqua L column and mobile phase in the gradient elution mode. The mobile phase consisted of 0.1% TCA in water and 0.1% TFA in water (70 : 30, v/v, component A) and consisted of 0.1% TCA in acetonitrile and 0.1% TFA in acetonitrile (64 : 36, v/v, component B) in a gradient elution: 0–30 min from 5% eluent B to 100% B; 30–45 min isocratic 100% B.

They were used, inter alia, the following additional chromatographic stationary phases Superdex 200 and XBridge Protein BEH C4 to confirm the purity of the standard. However, despite the manufacturer's efforts during the analysis of nesfatin-1 in several chromatographic systems, there were still two peaks of the standard.

Of course, we are aware that our results are not optimal due to the purity of the standard. However, we believe our preliminary results may be interesting for the readers.

The HPLC-DAD-FLD method showed satisfactory accuracy and precision for the analysis of nesfatin-1 in biological samples. The applied gradient elution programme allowed appropriate separation of the analyte from interferences under investigation in a single chromatographic run, as presented in Fig. 1.

Fig. 1.
Fig. 1.

Chromatogram nesfatin-1 standard (1 mg mL−1) with application of HPLC and diode array detector (DAD) at 275 nm

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01076

The calibration curves for the LOD and LOQ values were constructed by analyzing nesftain-1 standard in 0.1% trifluoroacetic acid (TFA) in water at five concentrations, from 0.1 to 1 mg mL−1, using two replicates. The calibration curves were obtained by means of the least square method.

The LODs and LOQs obtained for nesfatin-1 were calculated according to the formulas: LOD = 3.3 (SD/S) and LOQ = 10 (SD/S), where SD is the standard deviation of the response (peak area) and S is the slope of the calibration curve. The LODs and LOQs are demonstrated (Table 1).

Table 1.

Retention times and calibration data including calibration range, linear regression, R 2, SD of slopes and intercepts, instrumental limits of detection (LODs) and quantification (LOQs) obtained for Nesfatin-1 samples (0.1–1 mg mL−1) in six different wavelengths via HPLC-DAD-FLD

No. Detector tR (min) Calibration data
λ (nm) Range (mg mL−1) Linear regression R 2 SD of slopea SD of intercept LOD (mg mL−1) LOQ (mg mL−1)
1. DAD 8.112–9.755 275 0.1–1 y = 392.27x + 65.159 0.9371 71.84 41.31 0.35 1.05
2. DAD 8.111–9.755 280 0.1–1 y = 386.5x + 44.167 0.9578 57.37 32.99 0.28 0.85
3. FLD 8.100–9.807 200 0.1–1 y = 252.01x + 46.748 0.9733 29.50 16.96 0.22 0.67
4. FLD 8.098–9.805 230 0.1–1 y = 272.32x + 49.178 0.9729 32.17 18.50 0.22 0.68
5. FLD 8.456–9.804 240 0.1–1 y = 113.84x + 14.274 0.9562 17.23 9.91 0.29 0.87
6. FLD 8.453–8.885 250 0.1–1 y = 28.6x + 3.5226 0.9659 3.80 2.19 0.25 0.76

aSD of slope and intercept were obtained using the LINEST function (MS Excel 2010), which returns an array of the statistics for a calculated trend line by using the leastsquares method.

The detection of nesfatin-1 was compared using two different detection techniques. Obviously, due to the structure and properties of nesfatin-1, the use of a diode array detector is more expedient. A fluorescence detector (FLD) was also taken during the experiments. Due to the structure and properties of nesfatin-1, its detection with application of the fluorescence detector (FLD) is not satisfactory. However, it seems that after using derivatization of analyte, the use of a fluorescence detector (FLD) would be more appropriate.

To the best of our knowledge, this method is the first to combine the advantages of SPE on relatively new, commercially available columns with the Isolute Si-TsOH (SCX-3), as extraction technique with application of HPLC-DAD-FLD. This might be helpful in the biomonitoring of nesfatin-1 in human bovine samples.

Method validation is a one of principal aspect that is evaluated of the analytical procedures to increase the level of confidence. In this sense, the proposed method was validated based on the International Conference on Harmonization (ICH) guidelines in terms of linearity, selectivity, precision, accuracy, limit of detection and limit of quantitation [18]. Average recoveries of nesfatin-1 at optimal wavelengths with applications both detections techniques (DAD and FLD) after solid-phase extraction (SPE) is presented. Average recoveries of nesfatin-1 at five wavelengths with applications DAD and four wavelengths with applications FLD after solid-phase extraction on Isolute Si-TsOH SPE columns is presented. The Isolute Si-TsOH (SCX-3) is the bound equivalent of p-toluene sulfonic acid with pKa <1. This negatively charged sorbent (modified with a sulfonic acid group) retains basic (positively charged) analytes through strong cation exchange retention mechanism. The Isolute Si-TsOH SPE consists of the water wettable surface modified with a sulfonic acid functional group, therefore during determination of nesfatin-1 in fetal bovine serum samples which should be diluted with acidified solution such as 1% trifluoroacetic acid (TFA) in water. After loading the sample, nesfatin-1 is retained by ionic interactions – strong cation exchange interactions (Fig. 2).

Fig. 2.
Fig. 2.

Scheme: After loading the sample, nesfatin-1 has been retained by strong cation exchange (SCX) interactions on the Isolute Si-TsOH (SCX-3) SPE column. [Figure was created by the authors using the data provided by the producer of Isolute Si-TsOH (SCX-3) SPE columns.]

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01076

Whereas, the other interferences cannot enter the pores of the sorbent. When eluting the nesfatin-1 from the adsorbent, it is necessary to use an eluent with alkaline properties. The following mixture was used to elute the nesfastin-1: 5% ammonia with mixture of acetonitrile and water. The use of a mixture with ammonia allowed for appropriate elution conditions for the basic analyte from the sorbent with acidic properties. The use of an optimized SPE procedure allowed for slightly satisfactory obtaining of nesfatin-1 recovery value from biological sample (Table 2).

Table 2.

Mean recoveries (%) and relative standard deviations expressed as a percentage (RSD%) for nesfatin-1 extracted by SPE using Isolute Si-TsOH (SCX-3) SPE column

No. Detector λ (nm) Average Recovery (%)a RSD%a
1. DAD 275 53.6* 8
2. DAD 280 52.0* 8
3. FLD 200 55.4* 6
4. FLD 230 102.2** 5
5. FLD 240 119.6** 7
6. FLD 250 57.7* 6

aAverage recoveries (and RSDs) of the spiking fetal bovine serum samples at one level of nesfatin-1 (1 mg mL−1).

*Values of nesfatin-1 recoveries were reduced by the peak's areas of interferences (the area of the matrix peaks were subtracted).

**No matrix influence.

Average recoveries for nine different optimal wavelengths by the proposed SPE-HPLC-DAD-FLD method are given in Table 2. Method recovery was studied by analyzing twice replicates of samples spiked with nesfatin-1 at a concentration level of 1 mg L−1. Average recoveries from the spiked samples are shown in Table 2, with the RSD no larger than ±8.

It seems that the recovery values of nesfatin-1 could be higher after using SPE columns with a larger sorbent capacity than those used in this study.. Of course, the extraction conditions will be further optimized in the future.

The use of two different detection techniques enables more reliable identification of nesfatin-1 in biological samples. Two wavelengths of the DAD detector: 275 and 280 nm and four wavelengths of the FLD detector: 200, 230, 240 and 250 nm proved to be particularly useful for the identification of nesfatin-1 in biological samples (Fig. 3).

Fig. 3.
Fig. 3.

Comparison of following HPLC-FLD chromatograms at 250 nm: nesfatin-1 standard (top); chromatogram of blank fetal bovine serum matrix sample (middle); sample of blank fetal bovine serum matrix which was spiked with nesfatin-1 standard at 1 mg mL−1, after the SPE procedure with application of Isolute Si-TsOH SPE columns (bottom). FLD: a reinforcement of — 5 for top; a reinforcement — 7 for middle and for bottom

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01076

One of the most selective detectors used in liquid chromatography is the fluorescence detector (FLD). FLD is used for the determination of naturally fluorescing substances or related derivatives. The phenomenon of fluorescence occurs when an electron located at a higher energy level, due to the excitation of the molecule (e.g. by absorption of an energy quantum) it returns spontaneously to the previously occupied the orbit, emitting radiation at the same time.

The average time between the excitation of the molecule and the emission of radiation is 10−9 to 10−8 s. The determination of substances with FLD is therefore a measure the light it emits with a specific wavelength. Few compounds exhibit the natural ability to fluorescence, and therefore the particles of substances are excited with light of the wavelength that is absorbed by them. Due to the fact that chemical compounds absorb and emit light with a specific wavelength characteristic for them, FLD can demonstrate (especially for fluorescent compounds) sensitivity up to three orders of magnitude higher. The use of a fluorescence detector enables the correct identification of nesfatin-1 in samples without the need for additional purification. Optimizing the working conditions of the FLD detector makes it possible to properly estimate the value of nesfatin-1 recoveries in spiked samples.

One of the advantages of the FLD detector is the possibility to apply reinforcements ranging from 1 to 18, which allow the determination of low concentrations of analytes in different samples. It is possible to apply the enhancements only to the analyte peaks, without the enhancements of the matrix interferents. The use of reinforcements only for analytes (so-called ‘time windows’) greatly facilitates the analysis. Therefore, it is possible to determine lower and lower concentrations of analytes proportionally at higher and higher intensifications.

Of course, when determining nesphatin-1 in biological samples, we compared two different detection techniques. A fluorescence detector (FLD) could be used in laboratory practice to identify nesfatin-1 only after using derivatization and much greater reinforcements of the analyte signals (e.g. in the range 16–18 of FLD). The use of higher reinforcement with use FLD, while transforming nesfatin-1 into a compound with more fluorescing properties could allow the determination of concentrations in nanogram amounts per ml of biological sample. Therefore, it would be possible to determine also lower and lower concentrations of nesfatin-1 by using ever higher gain (reinforcement) values of the fluorescence detector.

The efficiency of the SPE procedure was evaluated using real fetal bovine serum samples. The procedure described for the determination of nesfatin-1 are inexpensive and can be applied to routine analysis of nesfatin-1 in human serum samples. Reinforcement 7 was applied throughout the analysis. The use of a higher reinforcements (e.g., 16–18), would allow the identification and quantification of nesfatin-1 at concentrations that would allow its determination directly in human fluids, e.g. plasma, serum, liquid cerebrospinal. The use of the so-called 'time windows' that allow only the analyte signals to be amplified (without the matrix interferences being amplified at the same time) may be applied.

Conclusions

To our knowledge, this is the first study of nesfatin-1 in biological samples with comparison of different detections techniques, such as DAD detection with much more sensitive FLD detection technique.

The conditions of the chromatographic analysis and the parameters of the FLD detector were optimized. Sufficient sensitivity on HPLC-FLD was achieved by applying reinforcements of described studies to nesfatin-1 (5 or 7 from range 1–18). The optimized conditions of analyses allow for the selective enhancement of analyte (from 6 min of analyse) in fetal bovine serum samples, which are separated from the remaining matrix interferences.

The HPLC-DAD-FLD separation after SPE of nesfatin-1 from interferences (which majority have been eluted before 6 min) is very effective because the nesfatin-1 can be identified and quantified. Moreover, low LOQs were attained in the range 0.67–1.05 mg mL−1, with a slight amplification of the analyte signal at 5 or 7.

During analyses with the FLD detector and using much greater reinforcement of the nesfatin-1 peak (three times higher, e.g. 15), it would be possible to detect nesfatin-1 in biological samples for analyte concentrations in nanograms per milliliter of sample.

To the best of our knowledge, this method is the first to combine the advantages of SPE on relatively new, commercially available columns with the Isolute Si-TsOH (SCX-3), as extraction technique with application of HPLC-DAD-FLD.

Recovery studies conducted at one spiking level of 1 mg mL−1 proved that the elaborated extraction procedures, especially SPE, support the possibility of nesfatin-1 residue determination in fetal bovine serum samples. This could aid in the identification and quantification of nesfatin-1 in human serum samples. The optimized procedure enables the identification of analyte in samples with a very small volume of 100 µL serum. The ability to identify and quantify nesfatin-1 in a volume of 100 µL of serum is particularly important when collecting serum samples from children, whose volumes are smaller compared to serum samples collected from adults. Therefore, the optimized procedure for the analysis of nesfatin-1 in small volumes of serum can be successfully used in the analysis of children's serum. The procedure can especially dedicated to the identification and quantification of nesfatin-1 for routine biomonitoring purposes of analyte in human serum samples. Both the SPE technique, chromatographic analysis with gradient elution mode and detection technique are fast and convenient. The experimental results presented in this paper are preliminary data. Due to the very high costs of purchasing the nesfatin-1 standard, they will be continued in the future also with the use of other analytical methods and detection techniques.

Funding

The following work was conducted as a part of a project entitled ‘Optimization of the conditions for the extraction, identification and quantification of nesfatin-1 in blood serum’ carried out from October 1, 2021 to June 30, 2022 within the financial support provided by the Medical University of Lublin, Poland (MINI GRANT 26/2021 – Student Research Group at the Department of Physical Chemistry, Chair of Chemistry, Faculty of Pharmacy, project manager Michał Szubartowski, scientific supervisor prof. dr. Tomasz Tuzimski).

Conflict of interest

The 1st author, Tomasz Tuzimski is a member of the Editorial Board of the journal. Therefore, the submission was handled by a different member of the editorial board, and he did not take part in the review process in any capacity.

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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s)

  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • A. Gumieniczek (Medical University of Lublin, Lublin, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • K. Karljiković Rajić (University of Belgrade, Belgrade, Serbia)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • Ł. Komsta (Medical University of Lublin, Lublin, Poland)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • Á. M. Móricz (Centre for Agricultural Research, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)
  • I.G. Zenkevich (St. Petersburg State University, St. Petersburg, Russian Federation)

 

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

Indexing and Abstracting Services:

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2021  
Web of Science  
Total Cites
WoS
652
Journal Impact Factor 2,011
Rank by Impact Factor Chemistry, Analytical 66/87
Impact Factor
without
Journal Self Cites
1,789
5 Year
Impact Factor
1,350
Journal Citation Indicator 0,40
Rank by Journal Citation Indicator Chemistry, Analytical 72/99
Scimago  
Scimago
H-index
29
Scimago
Journal Rank
0,27
Scimago Quartile Score Chemistry (miscellaneous) (Q3)
Scopus  
Scopus
Cite Score
2,8
Scopus
CIte Score Rank
General Chemistry 210/409 (Q3)
Scopus
SNIP
0,586

2020
 
Total Cites
650
WoS
Journal
Impact Factor
1,639
Rank by
Chemistry, Analytical 71/83 (Q4)
Impact Factor
 
Impact Factor
1,412
without
Journal Self Cites
5 Year
1,301
Impact Factor
Journal
0,34
Citation Indicator
 
Rank by Journal
Chemistry, Analytical 75/93 (Q4)
Citation Indicator
 
Citable
45
Items
Total
43
Articles
Total
2
Reviews
Scimago
28
H-index
Scimago
0,316
Journal Rank
Scimago
Chemistry (miscellaneous) Q3
Quartile Score
 
Scopus
393/181=2,2
Scite Score
 
Scopus
General Chemistry 215/398 (Q3)
Scite Score Rank
 
Scopus
0,560
SNIP
 
Days from
58
submission
 
to acceptance
 
Days from
68
acceptance
 
to publication
 
Acceptance
51%
Rate

2019  
Total Cites
WoS
495
Impact Factor 1,418
Impact Factor
without
Journal Self Cites
1,374
5 Year
Impact Factor
0,936
Immediacy
Index
0,460
Citable
Items
50
Total
Articles
50
Total
Reviews
0
Cited
Half-Life
6,2
Citing
Half-Life
8,3
Eigenfactor
Score
0,00048
Article Influence
Score
0,164
% Articles
in
Citable Items
100,00
Normalized
Eigenfactor
0,05895
Average
IF
Percentile
20,349
Scimago
H-index
26
Scimago
Journal Rank
0,255
Scopus
Scite Score
226/167=1,4
Scopus
Scite Score Rank
Chemistry (miscellaneous) 240/398 (Q3)
Scopus
SNIP
0,494
Acceptance
Rate
41%

 

Acta Chromatographica
Publication Model Online only
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Acta Chromatographica
Language English
Size A4
Year of
Foundation
1992
Volumes
per Year
1
Issues
per Year
4
Founder Institute of Chemistry, University of Silesia
Founder's
Address
PL-40-007 Katowice, Poland, Bankowa 12
Publisher Akadémiai Kiadó
Publisher's
Address
H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher
Chief Executive Officer, Akadémiai Kiadó
ISSN 2083-5736 (Online)

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