Name: simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures
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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).&#160; The authors thank Prof. Agnieszka &#346;cibior from the Laboratory of Oxidative Stress, Centre for Interdisciplinary Research, JPII CUL for sharing the equipment for cell culturing and protein quantification.&#160;

1. Preparation of standard 3NT, Kyn, 3HKyn, 3HAA, XA standard stock solutions
<ol>
	<li>Weigh the reagents in a vial with the highest accuracy (0.3 mg each). For better accuracy, scale up the reagents, adjusting the volume of the solvent according to step 1.2.</li>
	<li>Dissolve the reagents in 300 &#181;L of the solvent to obtain a stock solution of 1 g/L. Dissolve 3NT in 1% (v/v) formic acid (FA) in water; Kyn, 3HAA, and XA in dimethyl sulfoxide (DMSO); 3HKyn in water acidified to pH 2.5 with hydrochloric acid (HCl)....

<ol>
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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 an...

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&#8217;s, Huntington&#8217;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&#160;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, &#160;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. &#160;
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-&#947;)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&#160;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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1360px;" width="1359">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-hydroksy-DL-kynurenina</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">H1771</td>
			<td style="width:513px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-hydroxyanthranilic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">148776</td>
			<td>97%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-nitro-L-tyrosine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">N7389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Acetonitrile</td>
			<td style="width:175px;">Supelco</td>
			<td>1.00029</td>
			<td>hypergrade for LC-MS LiChrosolv</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Activated charcoal</td>
			<td style="width:175px;">Supelco</td>
			<td>05105</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Analytical balance</td>
			<td style="width:175px;">Ohaus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:411px;">Analytical column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>959764-902</td>
			<td>Zorbax Eclipse Plus C18 rapid resolution HT (2.1 x 100 mm, 1.8 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium formate</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">7022</td>
			<td>eluent additive for LC-MS, LiChropur, &#8805;99.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td style="width:175px;">Sigma</td>
			<td>1001887398</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caps for chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5185-5820</td>
			<td>blue screw caps,PTFE/red sil sepa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dish</td>
			<td style="width:175px;">Nest</td>
			<td>704004</td>
			<td>polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture plate</td>
			<td style="width:175px;">Biologix</td>
			<td>07-6012</td>
			<td>12-well, non-pyrogenic, non-cytoxic, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>HERAcell 150i Cu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5415R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-2278</td>
			<td>Eppendorf type, 1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-3693</td>
			<td>Falcon type, 50 mL, PP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic data acqusition and analysis software</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td></td>
			<td>LC/MSD ChemStation (B.04.03-S92)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic insert vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-1387</td>
			<td>100 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5182-0714</td>
			<td>2 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dimethyl sulfoxide</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">1.02950</td>
			<td>Uvasol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dubelcco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:175px;">PAN Biotech</td>
			<td>P04-41450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dual meter pH/conductivity</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td style="width:261px;"></td>
			<td>SevenMulti</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evaporator</td>
			<td style="width:175px;">Genevac</td>
			<td style="width:261px;"></td>
			<td>model EZ-2.3 Elite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Formic acid</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">5.33002</td>
			<td>for LC-MS LiChropur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glass bottle for reagents storage</td>
			<td style="width:175px;">Bionovo</td>
			<td>S-2070</td>
			<td>50 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Guard column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">959757-902</td>
			<td>Zorbax Eclipse Plus-C18 Narrow Bore Guard Column (2.1 x 12.5 mm, 5 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hydrochloric acid</td>
			<td style="width:175px;">Merck</td>
			<td>1.00317.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">L-kynurenine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">K8625</td>
			<td>&#8805;98% (HPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Magnetic stirrer</td>
			<td style="width:175px;">Wigo</td>
			<td style="width:261px;"></td>
			<td>ES 21 H</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microbalance</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td></td>
			<td>model XP6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Milli-Q system</td>
			<td style="width:175px;">Millipore</td>
			<td>ZRQSVPO30</td>
			<td>Direct-Q 3 UV with Pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quadrupole mass spectrometer</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>G1948B</td>
			<td>model 6120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:175px;">Sigma-Adrich</td>
			<td>048M4874V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td style="width:175px;">Bio Tek</td>
			<td></td>
			<td>Synergy 2 operated by Gen5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td style="width:175px;">Merck</td>
			<td>1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium dihydrogen phosphate</td>
			<td style="width:175px;">Merck</td>
			<td>1.05108.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Assay Dye Reagent Concentrate</td>
			<td style="width:175px;">BioRad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">See-saw rocker</td>
			<td style="width:175px;">Stuart</td>
			<td>SSL4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipette</td>
			<td style="width:175px;">Nest</td>
			<td>326001</td>
			<td>5 mL, polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td style="width:175px;">Merck</td>
			<td>1.06346.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent inlet filter</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5041-2168</td>
			<td>glass filter, 20 &#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6524</td>
			<td>1 L, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6526</td>
			<td>1 L, amber glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spreadsheet program</td>
			<td style="width:175px;">Microsoft Office</td>
			<td></td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stir bar</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-2003</td>
			<td>teflon coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for culture medium filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>7-8803</td>
			<td>regenerated cellulose, &#216; 30 mm, 0,45 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for mobile phase components filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-0018</td>
			<td>nylon, &#216; 30 mm, 0,22 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td style="width:175px;">VWR</td>
			<td>10062-900</td>
			<td>96-wells, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA Solution</td>
			<td style="width:175px;">Sigma-Aldrih</td>
			<td>T4049-100 mL</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:411px;">Ultra-High Performance Liquid Chromatography system</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">G1367D, G1379B, G1312B, G1316C</td>
			<td style="width:513px;">1200 Infinity system consisted of autosampler (G1367D), degasser (G1379B), binary pump (G1312B), column thermostat (G1316C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasound bath</td>
			<td>Polsonic</td>
			<td>104533</td>
			<td>model 6D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>Biosan</td>
			<td></td>
			<td>model V-1 plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xanthurenic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">D120804</td>
			<td>96%</td>
		</tr>
	</tbody>
</table>

simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures

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&#160;generates reliable results and can be further applied to other in vitro cellular models.

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 &#39;&#39;weak&#8221; point of the method. In our protocol, the ions are generated by electrospray ionization (ESI) in the positive mode, which was al...

Watch this Scientific Journal Video about Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures at JoVE.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

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 ...

Kynurenines are associated with immune response regulation in several diseases, including cancer. Reliable and validated methods for identifying multiple kynurenines aid in the development of more effective therapies. Our protocol uses liquid chromatography coupled with mass spectrometry to identify the different tryptophan metabolites generated by the kynurenine pathway in medium collected from cancer cell cultures.Unexperienced users may have some difficulty during the sample preparation or data acquisition and interpretation steps. By following our protocol and recommendations, one can avoid mistakes and obtain reliable data. To prepare stock solutions of the reagents, weigh out 0.3 milligrams of each reagent in a vial to the highest accuracy and dissolve each reagent in 300 microliters of the appropriate solvent to obtain one gram per liter stock solutions of each reagent.To prepare charcoal-treated culture medium, weigh out 280 milligrams of activated charcoal in a conical tube and add five milliliters of the liquid medium prepared for culturing the cells of interest. Shake the tube with the medium and charcoal on a seesaw rocker for two hours at room temperature and 50 oscillations per minute. At the end of the incubation, sediment the charcoal by centrifugation and carefully collect the supernatant without disturbing the charcoal, then filter the medium using a 0.45 micrometer syringe filter.To prepare the calibration solution, spike the charcoal-treated culture medium with 0.75 microliters of 0.1 gram per liter 3NT solution and add 3-H kynurenine, 3-HAA kynurenine, and xanthurenic acid to at least six different concentrations to cover all of the calibration ranges to a final volume of 150 microliters per sample. After vortexing, add 150 microliters of minus 20 degrees Celsius cold methanol supplemented with 1%formic acid to each tube for sample deproteinization and tightly cap and vortex the tubes again. After a 40-minute incubation at minus 20 degrees Celsius, centrifuge the samples and use an automatic pipette to transfer 270 microliters of each supernatant into individual flat bottom glass vials.Place the vials into an evaporator and use the appropriate program for water methanol fractions to gently evaporate the volatile components for about 30 minutes. When the tubes are dry, reconstitute each sample in 60 microliters of 0.1%formic acid in water and vortex the samples before transferring each solution into individual 1.5 milliliter tubes for centrifugation. Without disturbing the sediment, transfer the supernatants into chromatographic vials with conical glass inserts and place the vials into an LC-MS auto-sampler.Before running the samples, purge the LC system with the mobile phase to remove bubbles and to prime all of the solvent channels. Next, flush the guard and analytical column with 100%acetonitrile for about 30 minutes before flushing the system with 100%solvent A until a stable pressure is observed in the column, then set the appropriate LC parameters in the data acquisition software and construct a work list for running the samples on the LC system. To construct the calibration curve, in the data acquisition software, add the calibration standards into the work list and run the standards in triplicates, then integrate and measure the peak area corresponding to 3-hydroxykynurenine, kynurenine, 3-hydroxyanthranilic acid, 3-nitrotyrosine, and xanthurenic acid at the retention time of about 4.4, 10, 16, 21, and 30 minutes respectively.To collect supernatant samples from an in vitro cell culture, after 48 hours of standard cell culture of the cells of interest, transfer 500 microliters of supernatant from each well into individual 1.5 milliliter tubes and remove the cell debris by centrifugation, then transfer the medium supernatants into new tubes for minus 80 degrees Celsius storage until the analysis and maintain the cell pellets at minus 20 degrees Celsius until protein estimation analysis. To prepare samples for LC-MS analysis, transfer 149.25 microliters of each thawed culture medium sample into a new 1.5 microliter tube and add 0.75 microliters of 0.1 gram per liter 3NT solution to each tube. After preparing the samples as demonstrated, add 150 microliters of minus 20 degrees Celsius methanol supplemented with 1%formic acid to each tube and prepare the sample for LC-MS analysis as demonstrated.When the work list is complete, measure the peak area of each analyte and use an individual linear calibration equation dedicated for each analyte to calculate concentrations of the analytes present within each experimental sample. To assess cellular protein content corresponding to the sample of culture medium, resuspend each cell pellet in 100 microliters of PBS and freeze the samples at minus 20 degrees Celsius for one hour before thawing them on ice. After freeze-thawing the samples three times, collect the cell lysates by centrifugation and transfer the supernatants into new tubes without disturbing the pellets.Dilute the sample 10 times with fresh PBS and add the appropriate volumes of bovine serum albumin standard solution in duplicate to the appropriate wells of a 96-well plate. Load 10 to 15 microliters of each cell lysate sample supernatant to the appropriate wells of the 96-well plate and fill both the standard and sample wells to a final volume of 50 microliters of PBS per well. Next, add 200 microliters of a Bradford reagent diluted five times with ultra pure water to each well and incubate the plate for at least five minutes at room temperature.At the end of the incubation, load the plate onto a microplate reader and measure the absorbance at 595 nanometers, then construct a calibration curve to allow calculation of the amount of protein in each sample and divide the concentration of each kynurenine by the total protein content to normalize the kynurenine amount in each sample per one microgram of protein. Here are single quadrupole mass spectrometry results obtained during the analysis of medium containing 4.5 grams per liter of D-glucose and 10%fetal bovine serum. Note the small peak indicating the presence of kynurenine within the sample.Run a quality control sample before requiring the actual sample data to confirm the appropriate retention times of the analytes as a shift in LC signals or mismatches can be observed. Co-eluting matrix components can generate ions with the master charge ratio selected for the target analytes. For example, in this analysis, the peak from the unknown compound appeared in the scan period during which the ion of master charge ratio 206 was monitored.Due to the incompatibility of the retention time, the signal did not correspond to the analyte. Although the tryptophan isotope shared the same ion as master charge ratio 206, chromatographic analysis revealed that the tryptophan signal was well separated from the xanthurenic acid signal, indicating that the level of tryptophan present in the tested cancer cell medium did not impact xanthurenic acid quantification. This analysis of the culture medium from two types of cancer cell lines demonstrates that the evaluated human epithelial breast cancer cells released different amounts of tryptophan metabolites while the tested human ovarian cancer cells produced significantly higher levels of kynurenine.Using an internal standard during the sample preparation helps with reliable data acquisition. Normalization of the data to the protein content corrects for cell number variabilities within individual wells. Our protocol can be further expanded to determine additional analytes, but may accumulate in the culture medium under different experimental conditions.

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