Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.
The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method generates reliable results and can be further applied to other in vitro cellular models.
Kynurenine pathway (KP) is the major route of tryptophan (Trp) catabolism in human cells. Indoleamine-2,3-dioxygenase (IDO-1) in extrahepatic cells is the first and limiting enzyme of KP and converts Trp into N-formylkynurenine1. Further steps within KP generate other secondary metabolites, namely kynurenines that exhibit various biological properties. Kynurenine (Kyn) is the first stable Trp catabolite showing toxic properties and regulating cellular events after binding to the aryl hydrocarbon receptor (AhR)2. Subsequently, Kyn is transformed into several molecules either spontaneously or in the enzyme-mediated processes, generating such metabolites like 3-hydroxykynurenine (3HKyn), anthranilic acid (AA), 3-hydroxyanthranilic acid (3-HAA), kynurenic acid (Kyna), and xanthurenic acid (XA). Another downstream metabolite, 2-amino-3-carboxymuconic acid-6-semialdehyde (ACMS), undergoes non-enzymatic cyclization to quinolinic acid (QA) or picolinic acid (PA)1. Finally, QA is further transformed into nicotinamide-adenine dinucleotide (NAD+)3, the KP end-point metabolite that is an important enzyme cofactor. Some kynurenines have neuroprotective properties such as Kyna and PA, while the others, i.e., 3HAA and 3HKyn, are toxic4. Xanthurenic acid, which is formed from 3HKyn, presents antioxidant and vasorelaxation properties5. XA accumulates in aging lenses and leads to apoptosis of epithelial cells6. KP, described in the middle of the 20th century, gained more attention when its involvement in various disorders was demonstrated. Increased activity of this metabolic route and accumulation of some kynurenines modulate the immune response and are associated with different pathological conditions such as depression, schizophrenia, encephalopathy, HIV, dementia, amyotrophic lateral sclerosis, malaria, Alzheimer’s, Huntington’s disease, and cancer4,7. Some changes in Trp metabolism are observed in tumor microenvironments and cancer cells2,8. Moreover, kynurenines are considered as promising disease markers9. In cancer research, in vitro cell culture models are well established and widely used for preclinical evaluation of responses to anticancer drugs10. Trp metabolites are secreted by cells into the culture medium and can be measured to assess the status of the kynurenine pathway. Therefore, there is a need to develop appropriate methods for the simultaneous detection of as many KP metabolites as possible in a variety of biological specimens with an easy, flexible, and reliable protocol.
In this paper, we describe a protocol for the simultaneous determination of four kynurenine pathway metabolites: Kyn, 3HKyn, 3HAA, and XA by LC-SQ in a post-culture medium collected from cancer cells. In a modern analytical approach, liquid chromatography13,14,15,16 is preferred for the simultaneous detection and quantification of the individual tryptophan catabolites, in contrast to biochemical nonspecific assays utilizing Ehrlich reagent11,12. At present, there are many methods available for kynurenines determination in human specimens, mainly based on liquid chromatography with ultraviolet or fluorescence detectors13,17,18,19. Liquid chromatography coupled with a mass spectrometry detector (LC-MS) seems more suitable for this type of analysis, due to their higher sensitivity (lower limits of detection), selectivity and repeatability.
Trp metabolites have already been determined in human serum, plasma and urine13,20,21,22,23, however, the methods for other biological specimens, like cell culture medium are also desired. Previously, LC-MS was used for Trp-derived compounds in a medium collected after culturing of human glioma cells, monocytes, dendritic cells or astrocytes treated with interferon gamma (IFN-γ)24,25,26. Currently, there is a need for new validated protocols that can allow an assessment of several metabolites in different culture media, cells, and treatments used in cancer models.
The purpose of the developed method is to quantify (within one analytical run) four major kynurenines that can indicate abnormalities in KP. Presented here are critical steps of our recently published protocol for quantitative LC-SQ analysis of selected meaningful kynurenines using one internal standard (3-nitrotyrosine, 3NT) in the medium collected from in vitro cultured human cancer cells27. To our best knowledge, it is the first LC-SQ protocol for simultaneous quantification of 3HKyn, 3HAA, Kyn and XA in a culture medium obtained from the in vitro grown cells. Upon some modifications, the method might be further applied to study the changes in Trp metabolism in a broader range of cell culture models.
1. Preparation of standard 3NT, Kyn, 3HKyn, 3HAA, XA standard stock solutions
2. Preparation of charcoal treated culture medium
3. Making the calibration solutions and calibration curves
4. Preparation of quality control (QC) samples
5. Setting up the LC-MS system
6. Constructing the calibration curve
7. In vitro cell culture and sample collection for analysis
8. Prepare samples for LC-MS analysis
9. Assessing protein content and data normalization
LC-MS presents indisputable advantages in the quantification of biologically active molecules, even though some components of complex specimens cause so-called matrix effects and compromise ionization of analytes. It leads to ion suppression or ion enhancement, strongly decreasing accuracy and affecting a limit of detection/quantification of LC-MS, which is considered as a ''weak” point of the method. In our protocol, the ions are generated by electrospray ionization (ESI) in the positive mode, which was also employed in other studies on Trp metabolites determination13. ESI, however, is more prone to matrix effects than atmospheric pressure chemical ionization (APCI)28. Thus, for accurate quantification of Trp metabolites in a cell culture medium, we used an internal standard and matrix-matched calibration to compensate the matrix-dependent effects on the analyte signal and to correct the loss of the target compounds during a sample preparation step. We applied the same internal standard (3NT) for all the studied analytes (3HKyn, Kyn, 3HAA, XA). 3NT was not found in the original, fresh medium used for cell culturing and shows a similar behavior like target compounds under the applied analytical conditions. That makes 3NT the appropriate internal standard27. In addition, to simplify the protocol, in order to make it more accessible to a wide group of users, we propose only one step of sample preparation involving protein removal by treatment with methanol containing 1% (v/v) FA.
Within the presented protocol, it is recommended to prepare the calibration curves using the complete medium used for cell culture. However, the culture medium might contain endogenous Kyn that disturbs proper estimation of the analyte peak area, especially in the calibration solutions at low concentrations. Figure 1A illustrates LC-SQ results obtained during the analysis of DMEM containing 4.5 g/L D-glucose (DMEM-HG) and 10% (v/v) FBS used by our group for a cancer cell culture. We have found that the fresh complete culture medium initially contains some Kyn that can be removed by purification with activated charcoal (Figure 1B). While analyzing the experimental samples, the complete DMEM-HG culture medium was also analyzed as a control sample, and the Kyn peak area was subtracted from the peak area recorded for Kyn in each tested sample.
Figure 1: Representative LC-SQ results of complete DMEM-HG culture medium (A) before and (B) after pretreatment on activated charcoal. Samples of culture medium were spiked with the same amount of the internal standard (3NT). Please click here to view a larger version of this figure.
In the following step, a retention time for each target analyte was established (see a chromatogram in Figure 2A). The good practice in LC-MS analysis is to run a QC sample before the actual samples to confirm appropriate retention times of the analytes. Occasionally, a shift in LC signals can be observed, and mismatches tend to happen. Figure 2B shows a slight change in the retention times of the analytes, which, however, does not disturb correct measurements. On the other hand, Figure 2C presents a significant change in a position of the target peaks. As seen, 3NT signal was significantly shifted towards a shorter retention time and came out of the set time gate. In this case, an appropriate quantitative analysis was impossible because the internal standard signal cannot be measured correctly. This might be due to several factors, i.e. insufficient column equilibration, incorrect mixing of the solvents in the pump, use of inappropriate sample diluent or contamination of the column stationary phase. Figure 2D, however, represents a situation, when the registered peaks suffer from severely low intensities. This can be a result of an injection failure, a leak in the system or MS damage. Furthermore, the co-eluting matrix components can generate ions with m/z selected for the target analytes, like in Figure 3A, where the result from the analysis of MDA-MB-231 cells culture medium are shown. At the retention time of about 25 min, a strong signal derived from an unknown matrix component (compound X) was observed. The peak appears in the scan period, when the ion of m/z 206 (selected for XA) was monitored. However, this signal does not correspond to the analyte due to incompatibility of the retention time. To avoid mistakes during peak integration, a comparison of the retention time with that one recorded for QC samples is also recommended.
Figure 2. Examples of correct and incorrect results. The correct chromatograms of QC sample analysis at (A) medium and (B) low concentration levels recorded in different days. Example of the incorrect chromatogram resulting from significant change in the retention times of the analytes (C) and unsatisfactory intensities of peaks (D). Please click here to view a larger version of this figure.
Figure 3. Representative results of culture medium collected from different cancer cell lines. Culture medium from (A) MDA-MB-231 (human breast cancer) and (B) SK-OV-3 (human ovary cancer) cells was analyzed using LC-SQ. The compound X indicates the signal of an unknown compound present in the culture medium that co-elutes with the analyte and ionizes to form an ion with m/z selected for the analyte. Please click here to view a larger version of this figure.
The culture medium used for cancer cells (DMEM) contains significant amounts of Trp. Its isotope (13C-Trp) shares the same ion as XA (m/z 206) and might interfere with XA determination. However, under the applied chromatographic conditions, the signal from Trp is well separated from the XA signal. Therefore, we concluded that Trp present in DMEM does not affect XA quantification and accuracy of the method (Figure 4).
Figure 4. Position of tryptophan (Trp) and xanthurenic acid (XA) signals on MS chromatogram. Standard solutions of Trp (49.0 µmol/L) and XA (48.8 µmol/L) were prepared in Solvent A (20 mmol/L ammonium formate in water, pH 4.3) under optimized LC-SQ conditions. The ions of m/z 205 (corresponding to [Trp+H]+) and m/z 206 (corresponding to [XA+H]+) were monitored for Trp and XA, respectively. Please click here to view a larger version of this figure.
The presented analytical method successfully passed the validation test in terms of linearity, precision (coefficients of variation ≤15%), accuracy (96-104%), recovery (96-119%), and matrix effects for all analytes, like it was described in detail in our previous paper 27.
Finally, we confirmed an application of the described analytical approach to a medium collected from the in vitro cultured human cancer cells. Our data confirmed that a level of kynurenines can significantly vary in a culture medium collected from different cell types (Figure 3). We analyzed the culture medium from 2 types of cancer lines i.e. MDA-MB-231 and SK-OV-3 cells derived from breast and ovarian cancer, respectively. The selected lines are often used as models to study molecular events and for anti-cancer drug testing. As we observed, the cells cultured in a control standard medium released different amounts of tryptophan metabolites, i.e., MDA-MB-231 cells released a quantifiable amount of Kyn and XA at low nmol/L concentration (Figure 3A), while SK-OV-3 cells showed significantly more Kyn, very low secretion of XA, and no secretion of other studied kynurenines as indicated by the intensity of the corresponding peaks (Figure 3B).
This paper presents a detailed LC-SQ protocol for the simultaneous quantification of four major Trp metabolites (3HKyn, Kyn, 3HAA, XA) that are measured in a medium from in vitro cultured human cancer cells. Special attention is paid to the sample preparation, chromatographic/mass spectrometry procedure, and data interpretation, the most important points within the analysis.
In general, LC-MS analysis, due to high sensitivity, requires the highest standards of a protocol strictness and purity of used materials. It refers to an application of neatly cleaned and properly washed glassware, as well as high grade chemicals throughout the standard procedure. It is very important to use fresh water-based solvents of a mobile phase to avoid microbial contamination that in this sensitive approach might drastically affect the analysis. Before injecting into an LC system, the analyzed samples need to be carefully examined for the presence of any insoluble particles. It is important to note that the column should be sufficiently equilibrated before the sample analysis. Failure to comply with these rules can result in the contamination of the samples, the LC system, and the analytical column or in insufficient sample ionization in MS source, finally causing a shift of signals.
There are 2 key points within the protocol that need to be emphasized in order to get optimal results. The most critical part in the presented protocol is the sample preparation step. Using an inadequate procedure causes a loss of target compounds and consequently inaccurate quantification. In our approach, during the method development, sample preparation step was carefully optimized along with the selection of the solvent for protein removal. As we determined, sample treatment with methanol containing 1% (v/v) formic acid yielded the best recoveries for all studied kynurenines. The impact of a mobile phase composition on the extent of analyte ionization was also evaluated. We have checked mobile phases consisting of 0-20 mmol/L ammonium formate or ammonium acetate at different pH adjusted with formic or acetic acid. The mobile phase containing 20 mmol/L ammonium formate in water (pH 4.3, solvent A) and acetonitrile (solvent B) provided the best possible conditions for the simultaneous quantification of Kyn, 3HKyn, 3HAA and XA27. Another important step in LC-MS analysis is peak integration. Some kynurenines occur in a sample at very low concentration levels. In this case, the signals of co-eluting compounds might overlap with the target analyte signal or generate a peak at a similar retention time. An unexperienced user of this method may find some difficulties with a correct peak integration, as exemplified in Figure 2C. Thus, checking retention time for the analytes and comparing with a QC sample is required and highly recommended.
Good analytical practice includes selecting an appropriate internal standard for a quantitative analysis. Herein, we propose a simple and cost effective approach using only one internal standard (3NT) as an analogue of four kynurenines. The results confirmed that 3NT presents similar chromatographic behavior like the target compounds and in result it allows achieving a good precision and accuracy of the method27. Under the applied LC conditions, 3NT peak is well separated from the other analytes’ signals and easy to measure. We realize that isotopically labeled internal standards (ILIS) used in such analyses14,15 will provide better compensation of matrix effects and better control of all the variables that can lead to false results. On the other hand, ILISs for all target analytes are not always easily available, let alone their high cost. Also, ILISs are dedicated rather for LC-MS/MS than LC-SQ with Single Ion Monitoring (SIM) mode that we employed in this paper.
Using 3NT as an internal standard might be considered here as a limitation of the method due to a possibility of 3-nitrotyrosine formation on proteins29. However, in case of a culture medium, we did not observe any free endogenous 3-nitrotyrosine. It let us recognize 3NT as a suitable internal standard. However, we recommend a prior assessment of an initial endogenous 3NT level, especially when studying other cell types than tested in this report.
In summary, when selecting an analog of any analyte, one needs to consider many features, e.g., chemical similarity and initial absence in a sample matrix. Furthermore, a stability of the analogue internal standard in a sample matrix, its recovery and ionization efficiency in presence of matrix components might differ from the target compounds. Thus, all these features should be considered and carefully investigated during method development.
The analytical system in the presented method comprising an MS detector allowed us to achieve low limits of detection (0.0033 – 0.0108 µmol/L)27. This way simultaneous measurements of 3HKyn, Kyn, 3HAA, XA within the concentration ranges of 0.018 – 4.46 µmol/L, 0.0096 – 3.84 µmol/L, 0.033 – 13.06 µmol/L, and 0.019 – 4.87 µmol/L, respectively, was achieved. The main limitation of any LC-SQ analysis is associated with a proper separation of sample components on an LC column. Poor separation contributes to lower selectivity compared to the detection with a tandem quadrupole mass spectrometry utilizing product ions and Multiple Reaction Monitoring (MRM) mode. In order to avoid interferences from other matrix components that share the same or similar ions with a target molecule, the LC separation conditions must be carefully optimized. As an example, 13C Trp isotope (present in trace amounts) generates the same ion of m/z 206 as selected for XA monitoring. The misinterpretation was avoided here by optimizing the chromatographic conditions and satisfactory separation of Trp and XA eluted from the column (Figure 4).
In the future applications, some modifications to the protocol can be implemented. A potential user can modify a concentration of the internal standard (3NT) when the levels of studied kynurenines are expected to fall beyound the concentrations presented here. Importantly, 3NT concentration should be the same both in the calibration standards and in the experimental samples. In case of an extremely high analyte level, such as Kyn observed in SK-OV-3 cells, we recommend working with a higher concentration of 3NT. If sample dilution is required, it shoud be done at the final step of the protocol, before sample injection onto the LC column.
In the literature, there are some protocols proposed for LC-MS/MS quantitative analysis of kynurenines in a culture medium from different cells. One protocol provides a simultaneous determination of 3 metabolites, i.e., Kyn, 3HKyn and 3HAA but it is less sensitive (limit of quantifications (LOQ) at higher levels), in contrast to our method21. Another report presented a method that allows quantification of 4 kynurenines, including Kyn, 3HKyn, 3HAA and XA with similar LOQs25. None of them, however, was optimized for the detection of kynurenines generated by in vitro cultured cancer cells. The components of a sample matrix influence the analyte ionization in MS source by decreasing or increasing the signal, which is a cause of unreliable results. To obtain more accurate data, we proposed to use an analogue internal standard (3NT) in combination with a matrix-matched calibration for better compensation of matrix-dependent effects.
Using the presented methodology, we could observe that Kyn was the most abundant Trp metabolite in a culture medium collected from the analyzed cancer cell types, while the other studied metabolites under control culture conditions were not detected (excluding XA present in trace but detectable amounts). However, when the cells were stimulated with a potent immune activator – bacterial lipopolysaccharide, a larger amount of XA in addition to Kyn was secreted. On the other hand, the detected 3HKyn and 3HAA that are formed upstream of XA within KP were still under LOQ27. This suggests that some KP metabolites present in small amounts in a culture medium might be difficult to measure using the proposed LC-SQ approach. Nevertheless, the presented protocol is useful to identify changes in KP by quantification of the accumulating key Trp metabolites. Employing this methodology in future studies to engage an in vitro cell culture system will deliver novel biomarkers of immune-related diseases and cancer. Our approach brings a validated and reliable assay with relevant sensitivity for cellular models that are challenging due to low concentrations of the downstream KP metabolites. The provided instructions will allow researchers from different fields to utilize this approach for quantification of major kynurenines related to cancer and other diseases. Our data (unpublished) show that it is useful for studying KP modulation in cells exposed to different glycation products, but it also should find an application in other research fields and diseases with immune component, i.e., in endometrium biology and reproduction30.
The presented protocol can be further expanded to determine additional analytes that might accumulate in a culture medium during different experimental conditions. The appropriate reference standards of analytes should be employed for method validation in terms of accuracy, precision, linearity, recovery or selectivity before being used for quantitative analyses. Futhermore, depending on the research purpose, the protocol might be used for individual kynurenines (3HKyn, Kyn, 3HAA or XA). In this case, the ions corresponding to compounds that are not considered for analysis, might be removed from the MS settings. The suitable changes in the LC gradient program will help with cutting on analysis time.
When studying KP, it is relevant to assess the activity of IDO enzyme. This might be estimated simply by measuring Trp consumption and Kyn release into a culture medium or by calculating a kynurenine-to-tryptophan concentration ratio ([Kyn]/[Trp])31. In our approach, we have not measured a Trp concentration, however, the protocol might be expanded by adding this target in the LC-SQ analysis. As a more biochemically appropriate approach, we recommend to express IDO enzymatic activity as an amount of Kyn produced by cells per milligram of protein per minute as presented elsewhere32. We have noticed an advantage in using this approach in our other studies on cancer cells (manuscript in preparation) and recommend this method when using in vitro cell culture model.
The authors have nothing to disclose.
This work was supported by the European Union from European Regional Development Fund under the Operational Programme Development of Eastern Poland 2007-2013 (POPW.01.03.00-06-003/09-00), by Faculty of Biotechnology and Environmental Sciences of the John Paul II Catholic University of Lublin (Young Scientists grant for Ilona Sadok), Polish National Science Centre, OPUS13 (2017/25/B/NZ4/01198 for Magdalena Staniszewska, Principle Investigator). The authors thank Prof. Agnieszka Ścibior from the Laboratory of Oxidative Stress, Centre for Interdisciplinary Research, JPII CUL for sharing the equipment for cell culturing and protein quantification.
3-hydroksy-DL-kynurenina | Sigma-Aldrich | H1771 | |
3-hydroxyanthranilic acid | Sigma-Aldrich | 148776 | 97% |
3-nitro-L-tyrosine | Sigma-Aldrich | N7389 | |
Acetonitrile | Supelco | 1.00029 | hypergrade for LC-MS LiChrosolv |
Activated charcoal | Supelco | 05105 | powder |
Analytical balance | Ohaus | ||
Analytical column | Agilent Technologies | 959764-902 | Zorbax Eclipse Plus C18 rapid resolution HT (2.1 x 100 mm, 1.8 µm) |
Ammonium formate | Supelco | 7022 | eluent additive for LC-MS, LiChropur, ≥99.0% |
Bovine Serum Albumin | Sigma | 1001887398 | |
Caps for chromatographic vials | Agilent Technologies | 5185-5820 | blue screw caps,PTFE/red sil sepa |
Cell culture dish | Nest | 704004 | polystyrene, non-pyrogenic, sterile |
Cell culture plate | Biologix | 07-6012 | 12-well, non-pyrogenic, non-cytoxic, sterille |
Cell incubator | Thermo Fisher Scientific | HERAcell 150i Cu | |
Centrifuge | Eppendorf | model 5415R | |
Centrifuge | Eppendorf | model 5428 | |
Centrifuge tubes | Bionovo | B-2278 | Eppendorf type, 1.5 mL |
Centrifuge tubes | Bionovo | B-3693 | Falcon type, 50 mL, PP |
Chromatographic data acqusition and analysis software | Agilent Technologies | LC/MSD ChemStation (B.04.03-S92) | |
Chromatographic insert vials | Agilent Technologies | 9301-1387 | 100 µL |
Chromatographic vials | Agilent Technologies | 5182-0714 | 2 mL, clear glass |
Dimethyl sulfoxide | Supelco | 1.02950 | Uvasol |
Dubelcco’s Modified Eagle’s Medium (DMEM) | PAN Biotech | P04-41450 | |
Dual meter pH/conductivity | Mettler Toledo | SevenMulti | |
Evaporator | Genevac | model EZ-2.3 Elite | |
Fetal bovine serum | Sigma-Aldrich | F9665 | |
Formic acid | Supelco | 5.33002 | for LC-MS LiChropur |
Glass bottle for reagents storage | Bionovo | S-2070 | 50 mL, clear glass |
Guard column | Agilent Technologies | 959757-902 | Zorbax Eclipse Plus-C18 Narrow Bore Guard Column (2.1 x 12.5 mm, 5 µm) |
Hydrochloric acid | Merck | 1.00317.1000 | |
L-kynurenine | Sigma-Aldrich | K8625 | ≥98% (HPLC) |
Magnetic stirrer | Wigo | ES 21 H | |
Microbalance | Mettler Toledo | model XP6 | |
Milli-Q system | Millipore | ZRQSVPO30 | Direct-Q 3 UV with Pump |
Quadrupole mass spectrometer | Agilent Technologies | G1948B | model 6120 |
Penicillin-Streptomycin | Sigma-Adrich | 048M4874V | |
Plate reader | Bio Tek | Synergy 2 operated by Gen5 | |
Potassium chloride | Merck | 1.04936.1000 | |
Potassium dihydrogen phosphate | Merck | 1.05108.0500 | |
Protein Assay Dye Reagent Concentrate | BioRad | 500-0006 | |
See-saw rocker | Stuart | SSL4 | |
Serological pipette | Nest | 326001 | 5 mL, polystyrene, non-pyrogenic, sterile |
Sodium chloride | Sigma-Aldrich | 7647-14-5 | |
Sodium phosphate dibasic | Merck | 1.06346.1000 | |
Solvent inlet filter | Agilent Technologies | 5041-2168 | glass filter, 20 μm pore size |
Solvent reservoir (for LC-MS) | Agilent Technologies | 9301-6524 | 1 L, clear glass |
Solvent reservoir (for LC-MS) | Agilent Technologies | 9301-6526 | 1 L, amber glass |
Spreadsheet program | Microsoft Office | Microsoft Office Excel | |
Stir bar | Bionovo | 6-2003 | teflon coated |
Syringe filters for culture medium filtration | Bionovo | 7-8803 | regenerated cellulose, Ø 30 mm, 0,45 µm |
Syringe filters for mobile phase components filtration | Bionovo | 6-0018 | nylon, Ø 30 mm, 0,22 µm |
Tissue culture plates | VWR | 10062-900 | 96-wells, sterille |
Trypsin-EDTA Solution | Sigma-Aldrih | T4049-100 mL | |
Ultra-High Performance Liquid Chromatography system | Agilent Technologies | G1367D, G1379B, G1312B, G1316C | 1200 Infinity system consisted of autosampler (G1367D), degasser (G1379B), binary pump (G1312B), column thermostat (G1316C) |
Ultrasound bath | Polsonic | 104533 | model 6D |
Vortex | Biosan | model V-1 plus | |
Xanthurenic acid | Sigma-Aldrich | D120804 | 96% |