Abstract
Gut-derived uremic toxins contribute to the uremic syndrome and are gaining attention as potentially modifiable cardiovascular disease risk factors in patients with underlying chronic kidney disease. A simple, rapid, robust, accurate and precise ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of a panel of four gut-derived uremic toxins in human serum. The panel was comprised of kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate. Serum samples were protein precipitated with acetonitrile containing deuterated internal standards. Chromatographic separation of analytes was accomplished with an Acquity BEH C18 (2.1 × 100 mm, 1.7 μm) column by isocratic elution at a flow rate of 0.3 mL/min with a mobile phase composed of solvent A (10 mM ammonium formate; pH 4.3) and solvent B (acetonitrile) (85:15, v/v). Analytes were detected using heated electrospray ionization and selected reaction monitoring. The total run-time was 4 minutes. Standard curves were linear and correlation coefficients (r) were ≥0.997 for concentration ranges of 0.01–0.5 μg/mL for kynurenic acid, 0.25–80 μg/mL for p-cresol sulfate, and 0.2–80 μg/mL for hippuric acid and indoxyl sulfate. Intra- and inter-day accuracy and precision were within 19.3% for the LLOQs and ≤10.9% for all other quality controls. Matrix effect from serum was <15% and recovery was ≥81.3% for all analytes. The method utilizes a short run-time, simple/inexpensive sample processing, has passed FDA validation recommendations, and was successfully applied to study patients with kidney disease.
Keywords: kynurenic acid, hippuric acid, indoxyl sulfate, p-cresol sulfate, LC-MS/MS, uremic toxins
1. Introduction
Cardiovascular disease is the leading cause of death in patients with underlying chronic kidney disease (CKD) [1, 2]. Although the mechanism is unclear, gut-derived uremic toxins (GDUTs) accumulate in CKD and have been implicated in the acceleration of cardiovascular disease [3, 4]. Metabolism of tryptophan and tyrosine by gut microbes (i.e., the microbiota) leads to the generation of numerous uremic toxins, including kynurenic acid, hippuric acid, indoxyl sulfate and p-cresol sulfate [5]. Although more than 100 uremic toxins have been identified, this panel of four toxins was selected because they originate from gut metabolism, are representative of the uremic milieu, and remain persistently elevated despite treatment with hemodialysis [5–7]. Most importantly, each of these GDUTs is present in supraphysiologic concentrations in patients with kidney disease and has been independently associated with cardiovascular disease in humans [3, 8–10]. Thus, these four GDUTs are potential therapeutic targets to improve cardiovascular disease outcomes [11]. Measurement of GDUTs could be used to identify patients at risk for cardiovascular events, and to evaluate the efficacy of potential interventions to lower GDUT concentrations. Hence, simple, rapid, robust, accurate and precise analytical methods are needed to facilitate GDUT measurement and clinical-translational research in this area.
Several ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) methods for measuring kynurenic acid, hippuric acid, indoxyl sulfate or p-cresol sulfate in various combinations and/or with other analytes have been reported [12–21]. Each method has limitations in terms of total run-time, sample processing, overall complexity, calibration range, validation parameters assessed, and/or the choice of analytes simultaneously measured. For example, one of these methods has a 15 minute run-time and fairly labor-intensive sample processing [12], while another incorporates a complex LC-MS/MS detection approach with five segments requiring alternating polarity modes, co-elution of several analytes, an 8 minute total run-time, and expensive sample preparation [13]. Another multiple analyte method requires a total run-time of over 23 minutes using both positive and negative polarities, and the calibration range for indoxyl sulfate and p-cresol sulfate is not ideal for use in CKD patients [17].
The goal of this work was to develop and validate a simple, rapid, robust, accurate, and precise UPLC-MS/MS method to simultaneously measure kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate in human serum. The method was applied to a pilot study evaluating the efficacy of a novel therapeutic intervention to lower GDUTs in patients with Stage 3–4 CKD. Baseline data are presented that corroborate previously published data showing that these uremic toxins are markedly increased compared to healthy volunteers [12].
2. Material and Methods
2.1. Chemicals and reagents
Kynurenic acid, d5-kynurenic acid, hippuric acid, and indoxyl sulfate were purchased from Sigma (St. Louis, MO, USA). P-cresol sulfate, d7-p-cresol sulfate, d5-hippuric acid, and d4-indoxyl sulfate were purchased from Toronto Research Chemicals (North York, ON, Canada). Optima LC-MS grade methanol, acetonitrile, and water were purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultra-pure argon gas (>99.9%) was obtained from Matheson (Basking Ridge, NJ, USA). Ultra-pure nitrogen gas (>99.9%) was supplied from a nitrogen generator (Parker Balston, Haverhill, MA, USA). Double charcoal stripped, delipidized human serum was purchased from Golden West Diagnostics (Temecula, CA, USA). Oasis HLB cartridges (Waters, Milford, MA, USA) were used for the removal of residual kynurenic acid from stripped serum.
2.2. UPLC-MS/MS conditions and equipment
An Acquity UPLC system consisting of a binary solvent manager and a sample manager (Waters, Milford, MA, USA) was utilized. Chromatographic separation of analytes was achieved with an Acquity BEH C18 (2.1 × 100 mm, 1.7 μm) column fitted with an Acquity BEH C18 VanGuard pre-column (2.1 × 5 mm, 1.7 μm). The isocratic elution consisted of 85% solvent A (10 mM ammonium formate; pH 4.3) and 15% solvent B (acetonitrile). Formic acid was used to acidify solvent A to the target pH. The flow rate was 0.3 mL/min. The isocratic elution was selected to overcome limitations of gradient elution, like time for column equilibration to initial conditions. The column was held at 35°C, and the autosampler was kept at 10°C. Separation of analytes was achieved with a total run-time of 4 minutes.
Tandem mass spectrometric (MS/MS) detection was conducted with a TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA) fitted with a heated electrospray ionization (HESI) source. Detection of analytes was performed in positive and negative ionization modes by selected reaction monitoring (SRM). The polarity was switched from positive to negative mode at 1.4 minutes during the run. Spray voltages were set at 3000 V in positive polarity and 2000 V in negative polarity to prevent arcing. The vaporizer temperature and the ion transfer tube were set to 300°C. The collision gas pressure, auxiliary gas, and sheath gas and were set at 1.5 mTorr, 50 and 60 (arbitrary units), respectively. Other settings included a scan time of 0.05 seconds, scan width of 0.05 m/z, and full width at half maximum of 0.7 m/z for first and third quadrupole. Monitored ion transitions were m/z 190.1 → 144.0 for kynurenic acid (collision energy = 20V), m/z 195.1 → 149.1 for d5-kynurenic acid (collision energy = 18V), m/z 178.1 → 134.6 for hippuric acid (collision energy = 13V), m/z 182.9 → 139.6 for d5-hippuric acid (collision energy = 14V), m/z 212.0 → 80.4 for indoxyl sulfate (collision energy = 27V), m/z 215.9 → 80.4 for d4-indoxyl sulfate (collision energy = 31V), m/z 186.9→ 107.5 for p-cresol sulfate (collision energy = 26V), and m/z 193.9 → 114.6 for d7-p-cresol sulfate (collision energy = 24V). Data acquisition and processing was performed with Xcalibur software v4.0 (Thermo Scientific, San Jose, CA, USA).
2.3. Preparation of stock solutions, quality control (QC) samples, and calibration standards
Stock solutions containing d5-kynurenic acid, kynurenic acid, d5-hippuric acid, hippuric acid, d4-indoxyl sulfate, indoxyl sulfate, d7-p-cresol sulfate, and p-cresol sulfate were made in methanol. The analyte stocks were further diluted with methanol to make intermediate stocks, these were used to make serum calibration standards and QCs. Acetonitrile mixed with 0.075 μg/mL of d5-kynurenic acid, and 5 μg/mL of d4-indoxyl sulfate, d7-p-cresol sulfate, and d5-hippuric acid was used as a working internal standard solution. Double charcoal stripped human serum served as the matrix for calibrator and QC samples. Intermediate solutions were added into stripped serum to make calibration standards at concentrations of 0.010, 0.025, 0.050, 0.075, 0.10, and 0.50 μg/mL for kynurenic acid, 0.25, 1.0, 10, 20, 40, and 80 μg/mL for p-cresol sulfate, and 0.20, 0.50, 1.0, 10, 40, and 80 μg/mL for hippuric acid and indoxyl sulfate. These ranges were selected based on concentrations reported in the literature for all stages of CKD [4]. Four quality control samples (LLOQ, LQC, MQC, and HQC) were made by spiking stripped serum at concentrations of 0.010, 0.030, 0.080, and 0.400 μg/mL for kynurenic acid, 0.250, 0.750, 15, and 60 μg/mL for p-cresol sulfate, and 0.200, 0.600, 15, and 60 μg/mL for hippuric acid and indoxyl sulfate. All calibration standards and QC samples were stored at −80°C.
2.4. Sample processing
To begin sample preparation, 100 μL of internal standard solution was added to 50 μL of serum and briefly vortexed for 30 seconds. Following centrifugation (5 min at 10,000xg), the supernatant was transferred to a new micro-centrifuge tube, evaporated under nitrogen at 40°C and then reconstituted with 150 μL of water:acetonitrile (80:20, v/v) and vortexed for 30 seconds. A 20 μL aliquot was then injected onto the UPLC-MS/MS system. The dry-down and reconstitution steps were necessary to ensure sample compatibility with the mobile phase.
2.5. Validation of the assay
2.5.1. Calibration and linearity
A six point standard curve was constructed for each analyte. Lower limit of quantification (LLOQ) samples were processed in triplicate, and all other standards were processed in duplicate for three days. Analyte concentrations were quantified using their respective deuterated internal standards. Standard curves were constructed by graphing absolute peak-area ratios of analytes to internal standards versus the nominal analyte concentrations.
2.5.2. Accuracy and precision
Accuracy and precision were evaluated with twelve replicate LLOQ, LQC, MQC, and HQC samples on the first day, followed by six replicate LLOQ, LQC, MQC, and HQC samples on two separate days, totaling to n=24 samples at each QC level. All 24 QC samples were used to determine inter-day accuracy and precision, and the twelve replicates from the first day were used to determine intra-day accuracy and precision (Table 1). Accuracy (% bias) was based on the calculated mean concentration relative to the nominal analyte concentration. Precision was estimated by calculating relative standard deviation (% RSD) of the QC values.
Table 1.
Intra- and inter-day accuracy (%bias) and precision (RSD) for LLOQ, LQC, MQC and HQC
| Analyte | Level | Nominal conc. (μg/mL) | Intra-day | Inter-day | |||
|---|---|---|---|---|---|---|---|
| % Bias | RSD | % Bias | RSD | ||||
| Kynurenic acid | LLOQ | 0.01 | −5.29 | 13.3 | −5.47 | 10.8 | |
| LQC | 0.03 | 1.44 | 6.13 | −0.93 | 7.90 | ||
| MQC | 0.08 | 6.94 | 4.07 | 2.05 | 9.06 | ||
| HQC | 0.40 | 2.74 | 5.39 | −2.07 | 7.27 | ||
| Hippuric acid | LLOQ | 0.20 | −17.8 | 6.54 | −9.40 | 13.8 | |
| LQC | 0.60 | 1.61 | 6.59 | 0.70 | 6.55 | ||
| MQC | 15.0 | −7.04 | 3.44 | −6.15 | 4.48 | ||
| HQC | 60.0 | 3.12 | 2.81 | 1.20 | 4.16 | ||
| Indoxyl sulfate | LLOQ | 0.20 | −8.73 | 9.27 | −13.35 | 10.9 | |
| LQC | 0.60 | 10.9 | 2.11 | 9.50 | 5.29 | ||
| MQC | 15.0 | 3.08 | 5.46 | 1.10 | 5.29 | ||
| HQC | 60.0 | 2.11 | 6.05 | 3.30 | 5.51 | ||
| p-Cresol sulfate | LLOQ | 0.25 | −19.3 | 7.54 | −17.4 | 6.71 | |
| LQC | 0.75 | −0.50 | 3.10 | 0.40 | 3.46 | ||
| MQC | 15.0 | 3.30 | 1.57 | 2.80 | 1.77 | ||
| HQC | 60.0 | −3.70 | 1.99 | −5.30 | 2.74 | ||
2.5.3. Dilution integrity
Dilution integrity was also assessed. Samples were prepared by initially spiking stripped serum with each analyte to twice the concentration of the highest standards. Samples were then diluted 1:1, 1:2 and 1:4 with stripped serum prior to analysis. Triplicate dilution samples were processed. Accuracy and precision relative to the nominal values was determined for each dilution factor.
2.5.4. Stability
Analyte stability was determined with LQC and HQC samples subjected to various experimental conditions compared to freshly processed LQC and HQC samples. Stability (bias and RSD) was defined as being within 15% of the nominal freshly prepared QC sample concentrations. Stability samples were assessed in triplicate. Long-term stability was evaluated by analyzing samples that were stored for 2 months at −80°C. In order to assess the stability throughout sample processing, bench-top stability was investigated by leaving samples on the bench-top at room temperature for six hours before processing. Autosampler stability of processed samples was evaluated by injecting processed samples stored at 10°C for 24 hours and 72 hours. Finally, stability after freeze-thaw cycles was determined by subjecting samples to three 24-hour freeze/thaw cycles.
2.5.5. Matrix effect, recovery and carryover
Matrix effect of serum on kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate was assessed by comparing stripped serum spiked at the LQC, MQC and HQC concentrations listed in Table 2 to aqueous samples spiked at the same concentrations that were not extracted. Three replicate aqueous samples and three spiked samples of each QC level were analyzed using standard curves generated from serum-based standards. Matrix effect was calculated by comparing non-extracted aqueous QC samples to extracted serum-based QC samples, and was considered to be negligible if the measured concentration in serum deviated from aqueous samples by <15%.
Table 2.
Matrix effect of human serum on kynurenic acid, hippuric acid, indoxyl sulfate and p-cresol sulfate.
| Concentration (μg/mL) | Matrix Effect ( % ) | |||
|---|---|---|---|---|
| Nominal value | Aqueous Sample Mean ± SD | Human Serum Sample Mean ± SD | ||
| Kynurenic acid | 0.030 | 0.033 ± 0.001 | 0.028 ± 0.001 | 85.3 |
| 0.080 | 0.086 ± 0.000 | 0.080 ± 0.003 | 93.2 | |
| 0.400 | 0.411 ± 0.010 | 0.410 ± 0.014 | 99.7 | |
| Hippuric acid | 0.600 | 0.645 ± 0.057 | 0.609 ± 0.041 | 95.3 |
| 15.00 | 15.36 ± 1.138 | 14.89 ± 1.503 | 96.9 | |
| 60.00 | 62.18 ± 0.796 | 60.21 ± 2.092 | 96.8 | |
| Indoxyl sulfate | 0.600 | 0.622 ± 0.021 | 0.639 ± 0.022 | 103 |
| 15.00 | 15.38 ± 0.450 | 14.55 ± 0.542 | 94.7 | |
| 60.00 | 60.39 ± 3.222 | 59.44 ± 3.573 | 98.4 | |
| p-Cresol sulfate | 0.750 | 0.782 ± 0.026 | 0.752 ± 0.014 | 96.2 |
| 15.00 | 15.87 ± 0.693 | 15.87 ± 0.087 | 100 | |
| 60.00 | 61.43 ± 4.204 | 57.06 ± 0.622 | 93.1 | |
Recovery was assessed by comparing three replicates of extracted serum-based QC samples to blank serum samples spiked after the extraction procedure. Measured concentrations of non-extracted aqueous, and the post-extraction spiked blank serum QC samples were defined as 100%. Means, standard deviations, and coefficients of variation were determined. Carryover was also assessed by randomly injecting at least six blank injections of 80:20 water:acetonitrile (v/v) (mobile phase composition) during the analytical sequence.
2.6. Application of the method
The method was applied to a clinical study evaluating the efficacy of a novel therapeutic intervention to lower GDUTs in patients with CKD. The University of Pittsburgh Institutional Review Board approved the protocol, and all participants provided written informed consent. Serum samples were collected and stored at −80°C until analysis.
3. Results and Discussion
We aimed to develop and validate a simple, rapid, and robust UPLC-MS/MS method for simultaneously measuring kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate in human serum to support studies evaluating the efficacy of novel therapeutic interventions to lower GDUTs in patients with CKD. The method was comprehensively validated according to the 2018 U.S. FDA guidance for bioanalytical method validation [22].
3.1. Chromatographic separation
Representative chromatograms showing separation of kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate obtained from the analysis of serum samples obtained from a CKD patient and a healthy volunteer are shown in Figure 1. Analyte retention times were 1.32, 1.52, 2.11, and 3.30 minutes for kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate, respectively. All peaks were narrow and well-separated with baseline resolution. The human serum samples had comparable chromatography to all other tested samples, demonstrating no observable interference. The MS/MS spectra of the product ion scans and compound structures are presented in Figure 2. The most intense fragment ion for each analyte and the corresponding fragment in internal standards was selected for SRM. This allowed for selective and sensitive measurement of analytes.
Fig. 1.
Representative chromatograms obtained from the analysis of a kidney disease patient at the baseline study visit and a healthy volunteer. (A) Concentrations observed in the kidney disease patient were kynurenic acid 0.19 μg/mL, hippuric acid 1.89 μg/mL, indoxyl sulfate 4.73 μg/mL, and p-cresol sulfate 22.2 μg/mL. (B) Concentrations observed in the healthy volunteer were kynurenic acid 0.05 μg/mL, hippuric acid 0.38 μg/mL, indoxyl sulfate 0.25 μg/mL, and p-cresol sulfate 0.29 μg/mL. Abbreviations: KA, kynurenic acid; d5-KA, d5-kynurenic acid; HA, hippuric acid; d5-HA, d5-hippuric acid; IS, indoxyl sulfate; d4-IS, d4-indoxyl sulfate; PCS, p-cresol sulfate; d7-PCS, d7-p-cresol sulfate.
Fig. 2.
MS-MS Spectra acquired on the TSQ Quantum Access MAX triple quadrupole mass spectrometer in positive and negative mode. All respective structures of parent compounds are embedded in the mass spectras. Abbreviations: KA, kynurenic acid; d5-KA, d5-kynurenic acid; HA, hippuric acid; d5-HA, d5-hippuric acid; IS, indoxyl sulfate; d4-IS, d4-indoxyl sulfate; PCS, p-cresol sulfate; d7-PCS, d7-p-cresol sulfate.
3.2. Validation of the assay
3.2.1. Calibration and linearity
Linear standard curves were achieved at 0.01–0.5 μg/mL for kynurenic acid, 0.25–80 μg/mL for p-cresol sulfate, and 0.2–80 μg/mL for hippuric acid and indoxyl sulfate. The correlation coefficient (r) was 0.997 or greater for all analytes. Standard curves were calculated for each analyte using weighted (1/x) linear regression analysis. LLOQ standards for each calibration curve exhibited excellent intra- and inter-day accuracy and precision (bias and RSD were within 18.9% and 12.6%, respectively), and signal-to-noise ratios were above 10:1 for all analytes. Intra- and inter-day accuracy and precision were within 12.3% for other standard levels.
3.2.2. Accuracy and precision
Intra-day and inter-day accuracy (% bias) and precision (% RSD) were determined at four levels according to the most recent FDA guidelines: the three quality controls (LQC, MQC, and HQC) and the LLOQ [22]. Assay bias for LLOQ samples was within 19.3%, while the RSD was within 13.8% (Table 1). Assay biases for all QC samples were within 10.9%, while the RSD was within 9.06% (Table 1). These values met the FDA recommendations for acceptable accuracy and precision being within 20% and 15% for LLOQs and other QCs, respectively [22].
3.2.3. Dilution integrity
Dilution integrity was assessed to determine if samples that are above the standard curve ranges can be diluted and accurately quantified. Dilution analysis exhibited excellent accuracy and precision, with bias and RSD within 11.04% and 6.19%, respectively.
3.2.4. Matrix effect, recovery, and carryover
Matrix effect from serum was negligible (<15%) for all analytes over three different concentrations tested (Table 2). Recovery was acceptable ranging from 81.3% to 106% for the LQC, MQC and HQC concentrations (Table 3). Carryover was negligible using at least six blank injections of mobile phase randomly throughout the sequence. No additional washes or column flushing was necessary, which is one advantage of the current assay over similar methods [12].
Table 3.
Recovery of kynurenic acid, hippuric acid, indoxyl sulfate and p-cresol sulfate in human serum.
| Concentration (μg/mL) | Recovery ( % ) | |||
|---|---|---|---|---|
| Nominal value | Post-extraction Mean ± SD | Extracted Mean ± SD | ||
| Kynurenic acid | 0.030 | 0.035 ± 0.001 | 0.031 ± 0.002 | 90.0 |
| 0.080 | 0.100 ± 0.000 | 0.081 ± 0.004 | 81.3 | |
| 0.400 | 0.451 ± 0.005 | 0.414± 0.006 | 91.8 | |
| Hippuric acid | 0.600 | 0.631 ± 0.037 | 0.623 ± 0.058 | 98.8 |
| 15.00 | 14.80 ± 0.895 | 13.24 ± 0.174 | 89.6 | |
| 60.00 | 53.93 ± 1.598 | 57.03 ± 1.806 | 106 | |
| Indoxyl sulfate | 0.600 | 0.696 ± 0.012 | 0.835 ± 0.086 | 106 |
| 15.00 | 16.82 ± 0.450 | 16.70 ± 0.706 | 99.3 | |
| 60.00 | 60.38 ± 0.786 | 58.82 ± 6.336 | 97.4 | |
| p-Cresol sulfate | 0.750 | 0.895 ± 0.012 | 0.831 ± 0.021 | 92.8 |
| 15.00 | 17.56 ± 0.077 | 15.40 ± 0.178 | 87.7 | |
| 60.00 | 67.06 ± 0.250 | 59.76 ± 0.558 | 89.1 | |
3.2.5. Stability
Long-term, bench-top, autosampler, and freeze-thaw stability were evaluated for analytes at LQC and HQC levels as shown in Table 4. Samples were stable for at least 6 hours on the bench-top, and at least 72-hours in the autosampler. Three 24-hour freeze-thaw cycles also did not impact analyte stability. All stability samples were within 15% of the freshly processed samples, demonstrating suitable stability at all conditions tested.
Table 4.
Stability of kynurenic acid, hippuric acid, indoxyl sulfate and p-cresol sulfate QCs (LQC and HQC).
| Analyte | Target (μg/mL) | Stability (% remaining) # | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Benchtop (6 h) | Autosampler (72 h) | Freeze/Thaw (3 cycles) | Long-term Storage (60 days) | ||||||
| % of Target | % RSD | % of Target | % RSD | % of Target | % RSD | % of Target | % RSD | ||
| Kynurenic Acid | 0.030 | 91.1 | 1.9 | 86.6 | 0.43 | 100.1 | 6.6 | 105.4 | 6.9 |
| 0.400 | 92.1 | 1.6 | 89.9 | 4.2 | 95.2 | 12.6 | 103.7 | 13.7 | |
| Hippuric Acid | 0.600 | 107.1 | 8.1 | 102.4 | 7.8 | 92.6 | 2.2 | 88.6 | 2.1 |
| 60.00 | 99.9 | 5.5 | 104.6 i | 5.3 | 99.8 | 7.2 | 103.3 | 7.5 | |
| Indoxyl Sulfate | 0.600 | 96.5 | 4.5 | 98.4 | 2.9 | 98.8 | 3.6 | 98.8 | 3.6 |
| 60.00 | 98.2 | 4.1 | 108.9 | 3.3 | 106.3 | 5.4 | 99.9 | 1.7 | |
| P-Cresol Sulfate | 0.750 | 102.3 | 2.9 | 104.6 | 4.1 | 97.3 | 1.7 | 97.5 | 1.7 |
| 60.00 | 97.6 | 0.9 | 100.9 | 1.0 | 103.2 | 5.8 | 101.1 | 2.0 | |
n = 3 for LQC and HQC samples
3.3. Method application
Kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate concentrations were measured in healthy volunteers and Stage 3–4 CKD patients. Estimated glomerular filtration rate (eGFR) was used to define healthy volunteers, Stages 3a, 3b, and 4, as eGFR values of >120, 59–45, 44–30, and 29–15 mL/min/1.73 m2, respectively. Representative chromatograms obtained from analysis of a Stage 3b CKD patient with an eGFR of 32 mL/min/1.73 m2 and a healthy volunteer are presented in Fig. 1A. and 1B., respectively. Stepwise increases were observed in all GDUTs as CKD advanced compared to healthy volunteers. These increases were the most notable for indoxyl sulfate and p-cresol sulfate. For example, as shown in Fig. 1, the baseline serum indoxyl sulfate concentration was 18-fold higher in the CKD patient, at 4.73 μg/mL compared to 0.25 μg/mL in the healthy volunteer. Similarly, the baseline p-cresol sulfate concentration in the CKD patient was 75-fold higher, at 22.2 μg/mL compared to 0.29 μg/mL. These data suggest that GDUTs begin accumulating to high concentrations long before complete loss of kidney function (i.e., diagnosis of end-stage kidney disease). The extent of GDUT accumulation is alarming because each of these toxins is associated with and may accelerate cardiovascular disease [3, 8–10]. As such, therapeutic strategies to reduce GDUTs might decrease cardiovascular disease outcomes, especially in these earlier stages of CKD. Measurement of the GDUT panel described here may facilitate clinical investigations of the impact of lowering GDUTs on cardiovascular disease progression.
4. Conclusions
A simple, rapid and robust UPLC-MS/MS method for the simultaneous determination of kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate in human serum was developed and validated. The assay has excellent accuracy, precision, and recovery. Additional strengths include a four-minute UPLC run-time, simple/inexpensive sample processing, and meeting contemporary FDA validation requirements. The method was successfully applied to a clinical study evaluating the efficacy of a novel therapeutic intervention to lower GDUTs in patients with CKD.
Highlights.
UHPLC-MS/MS method for measurement of gut-derived uremic toxins in human serum.
The method is validated with excellent recovery, accuracy and precision.
The method is robust with a short 4 minute run-time
The method was applied to a kidney disease patient and a healthy volunteer.
Funding
This work was supported in-part by the National Institutes of Health grants TL1TR001858 [AJP], R01-GM107122 [TDN], and R21-DK108093 [JRS, TDN]. AJP was also supported in-part by a fellowship from the American Foundation for Pharmaceutical Education
Footnotes
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