Simultaneous Analysis of Glucuronyl- and Sulpho-Conjugates of Intestinal Putrefactive Compounds in Human Urine by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry

Glucuronyland sulpho-conjugates of intestinal putrefactive compounds (IPC) are considered kidney disease-associated toxic agents. Recently, several simultaneous methods of detection by liquid chromatography tandem mass spectrometry (LCMS/MS) of the conjugated forms of glucuronyland sulpho-conjugates in blood serum were reported. To analyze these conjugated forms in urine, we modified and validated the LC-MS/MS detection methods. The range of the calibration curves were determined using phenyl glucuronide (PhG; 1.56-25.0 μM), indoxyl glucuronide (IndG; 1.25-20.0 μM), p-cresyl glucuronide (PCG; 0.625-10.0 μM), phenyl sulfate (PhS; 3.13-50.0 μM), indoxyl sulfate (IndS; 7.81-250 μM) and p-cresyl sulfate (PCS; 1.56-25.0 μM). Urine was diluted 2-4 fold to determine PhG, IndG and PCG concentrations, and 4-16 fold to determine PhS, IndS and PCS concentrations. We validated the newly-modified detection methods by a recovery test, and intra-day and inter-day repeatability. We analyzed six clinical urine samples, PCS and IndS, major toxic agents, which were mostly detected. Creatinine-corrected values of PCS and IndS were high in six conjugates. In summary, in the present work, the concentrations of six urinary conjugated forms of IPC were successfully measured by LC-MS/MS.


Introduction
The hindgut microbiota produces many types of metabolites such as short-chain fatty acids, hydrogen, methane, hydrogen sulfide, ammonia, amines and phenols via anaerobic fermentation [1]. Short-chain fatty acids (SCFA) are the major and most useful metabolites produced during hindgut fermentation. SCFA are used as metabolic energy when they are absorbed by the host [2]. Moreover, during hindgut fermentation, intestinal putrefactive compounds (IPC) such as phenol, indole and p-cresol, which are considered toxic metabolites, are often produced and absorbed from the lumen into blood. Phenol, indole and pcresol are almost entirely metabolized to glucuronyl-or sulpho-conjugates in the intestinal wall and liver [3].
The kidneys possess an excretory function of endogenous and exogenous substances. The conjugated forms of IPC are among those substances excreted in the urine by the kidneys. Chronic kidney disease (CKD), mainly caused by chronic nephritis and metabolic syndromes such as hypertension and diabetes, increases proteinuria and decreases kidney function.
Recently, research on the toxicity of the conjugated forms of IPC has increased exponentially [4]. In particular, the relation of the gut microbiota and CKD has attracted attention [5]. For example, the conjugated forms of IPC were observed to accelerate CKD [6]. Among the conjugated forms of IPC, p-cresyl sulfate (PCS) and indoxyl sulfate (IndS) are representative and have been repeatedly associated with overall mortality, cardiovascular disease and progression of CKD [7]. Particularly, the progression of CKD caused by IndS is believed to be induced by gene transcription associated with renal fibrosis [6]. IndS increases extracellular superoxide dismutase-sensitive O 2 production and intracellular hydroxyl radical production in mesangial cells [8]. In addition, showing a mechanism similar to that of IndS [9], PCS is also a toxic factor in CKD progression [10,11]. Rapid and accurate detection of the conjugated forms of IPC in urine and blood is likely to be useful for establishing the occurrence or degree of progression of CKD. Several simultaneous methods by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of quantification of the conjugated forms of IPC in blood serum were reported [7,12]. However, those methods were designed for the analysis of human serum, and their detection sensitivity of the conjugated forms of IPC in human urine is still limited.
In the present study, we modified the previously described determination methods [7] to analyze human urine samples by ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). In addition, we validated the newly-modified methods.
Methanol and purified water of MS grade were purchased from FUJIFILM-Wako Pure Chemical Corporation (Osaka, Japan). Other reagents used in this study were purchased from Sigma-Aldrich Japan.

Preparation of stock solutions and working solution
The stock solution of all standards was separately prepared using the MS-grade purified water. The stock concentrations of PhG, PhS, IndS and PCS were 10.0 mmol/L, whereas those of IndG and PCG were 1.00 mmol/L. The internal standard solution (1 mL) was prepared by adding 0.1 mg of phenylacetyl-d5 L-glutamine, 0.1 mg of p-cresyl sulfate-d7, and 0.8 mg of indoxyl sulfate-d6 to water. The stock solutions were kept at -80˚C until use.

Samples of urine
Six urine samples were purchased from KAC Co. Ltd. (Kyoto, Japan). The age and sex of donors were 22-47 years and Western female/male: 3/3, respectively. All samples were collected from donors to receive informed consent with the ethical aspect by a commercial supplier (Clinical Trials Laboratory Services Ltd., London, UK). Urine samples were stored at -80 ˚C until use.

Preparation of blank urine sample
The blank urine sample was prepared from the aforementioned urine samples. All frozen samples were thawed to room temperature and equally mixed. To remove sulpho-conjugates, the mixed urine sample was transferred to Oasis WAX cartridges (Waters, Milford, MA, USA). To remove glucuronyl-conjugates, the filtrated urine was then transferred to Oasis Hydrophilic-Lipophilic cartridges (Waters). These cartridges were conditioned with purified water prior to usage. The double-filtrated urine was used as the blank urine sample.

Instruments
The analytical condition was adjusted as previously described [7], with some modifications. Briefly, an UHPLC device equipped with a binary solvent manager, an autosampler, a column heater, and tandem mass spectrometry (Acquity TQD System; Waters) was used to detect the conjugated forms of IPC. Chromatographic separation was conducted using an ACQUITY CSH Fluoro-Phenyl column 2.1φ×50 mm (particle size 1.7 μm; Waters) with a guard column (ACQUITY CSH Fluoro-Phenyl 2.1φ×5 mm VanGuard; particle size 1.7 μm; Waters). The mobile phase, delivered at a flow rate of 0.5 mL/min at 40 ˚C, was a gradient of 0.1% formic acid as solution A and MS grade methanol as solution B.
The gradient procedure was as follows: start point at 3% solution B and a gradually increase to 16% in 1 min, then to 80% in 3 min, to 95% in 30 s, maintained 95% for 1 min, then to 3% in 30 s, and maintained 3% for 2.5 min. Total run time of the method was 8.5 min.
Solutes were detected using a TQD System with a Z-spray ion interface. The system was controlled using Waters MassLynx mass spectrometry software (Waters). Ionization was carried out using an alternating electrospray negative ionization mode. The negative ion mode was set as follows: ion source temperature, 150 ˚C and capillary desolvation temperature, 500 ˚C. The multiple-reaction monitoring (MRM) transitions, cone voltage and collision energy were individually optimized for each compound and the optimal dwell time was experimentally determined for each component ( Table 1). The injection volume was 5 µL.

Method validation
Validation was conformed to the guidelines for analytical procedures and methods validation for analytical procedures and methods validation by International Conference on Harmonisation (ICH) [13] and Food and Drug Administration (FDA) [14].

Linearity of calibration curve
The working standard solutions were prepared from the stock solutions and diluted 2-, 4-, 8-, 16-, 32-, 64-or 128fold with the blank urine sample. To evaluate the linearity of calibration curve, three replicates of working standard solutions were prepared and loaded for UHPLC-MS/MS analysis on a same day. The peak area values of each calibration standard were divided by the peak area of the respective internal standard and plotted afterward. Calibration curves were constructed with 5-6 plots using linear regression and the coefficient of determination was then calculated according to the ICH guideline [13]. To validate the linearity of calibration curve, values higher than 0.995 of the mean coefficient of determination (r 2 ) of three replicates were determined to be acceptable. The limit of quantitation (LOQ) concentration was determined by the criteria such as "the lowest values within 80-120% against theoretical value".

Intra-day and inter-day repeatability
Intra-day repeatability for accuracy and precision were determined by the values of three replicates of working solutions obtained in a day. Accuracy was defined as follows: = (the mean measured value of 3 replicates) / (the theoretical value)  100. Precision was defined as follows: = (the standard deviation of 3 replicates) / (the mean measured value)  100. Values of accuracy and precision were determined to be acceptable when they were 85 to 115%, and lower than 15%, respectively [at LOQ, 80 to 120% and lower than 20%, respectively].
Inter-day repeatability for accuracy were determined by the values of three replicates of working solutions obtained for consecutive 3 days. Inter-day repeatability for accuracy and precision was defined using the same calculation procedure as for intra-day repeatability.

Carryover test
A carryover test was conducted as follows: the highest concentration of a given working standard solution (PhG, 50.0 μmol/L; IndG and PCG, 20.0 μmol/L; PhS, 200 μmol/L; IndS, 500 μmol/L; PCS, 50.0 μmol/L) was injected, followed by 3 times of an injection of the blank urine. Appearance of peaks of standard substances of 6 conjugates and internal standards were recorded during the loading of the blank urine.

Validation of dilution
To validate the dilution, the aforementioned mixed urine was diluted 2-, 4-, 8-or 16-fold with blank urine. All samples were prepared in triplicate. Internal standards were added to both diluent, blank urine and non-diluted urine, performed sample preparation as mentioned chapter 2.5. and their peak areas were measured. Relative peak area ratios of diluents and non-diluted urine against the blank urine were calculated. A relative peak ratio was considered acceptable if the value was 85 to 115%. Next, the precision of the concentrations of the conjugated forms within the diluents was determined. In addition, concentration ratios of diluents against 2-fold diluents in glucuronyl-conjugates such as PhG, IndG and PCG and 4-fold diluents in sulpho-conjugates such as PhS, IndS and PCS were calculated. The values of the concentration ratio and precision were considered to be acceptable if these values were 85 to 115% and lower than 15%, respectively.

Recovery test
Three (low, middle and high) different concentrations from each standard substance (Table 2) were prepared. Six clinical urine samples (chapter 2.3.) were equally mixed and diluted 4-fold or 16-fold with blank urine. Ten µL of the standard solutions was added to 190 µL of urine diluted with blank urine at three levels (low, middle or high). They were named Solution X-4low, Solution X-4middle, Solution X-4high, Solution X-16low, Solution X-16middle, or Solution X-16high. One hundred and ninety µL of 4 or 16-fold diluted urine was diluted with 10 µL of blank urine and the resulting solutions were named Solution Y-4 or Solution Y-16. In addition, Solution Z-low, Solution Z-middle or Solution Z-high were prepared by mixing 190 µL of blank urine with 10 µL of each standard solution ( Table 2). The recovery ratio of each substance was defined as follows: = (the concentration of 6 conjugates in Solution Xthe concentration of 6 conjugates in Solution Y) / (the concentration of 6 conjugates in Solution Z)  100. A recovery test was considered successful at 70-110%. The recovery test was carried out in triplicate.

Freeze-thaw stability
Freeze-thaw stability was evaluated with the aforementioned diluted urine using in chapter 2.7.5. Nine volumes of 4-fold diluted urine and nine volumes of 16-fold diluted urine (120 µL) were prepared. Six volumes of each diluent were frozen at -80 ˚C and stored overnight. The remaining three volumes of each diluent were used as unfrozen control. On the next day, all frozen volumes were thawed to room temperature, and three volumes of each diluent were re-frozen at -80 ˚C. Three-freeze-thaw cycle testing was carried out on re-frozen volumes. The remaining three volumes of each diluent were used as one-time freezethaw samples. Post-treated urine samples were analyzed by UHPLC-MS/MS and the peaks were detected. The freezethaw stability ratio was defined as follows: = (no freezing control) / freeze-thaw sample)  100.

Measurement of clinical urine sample
Clinical validation of the six urine samples (chapter 2.3.)   was carried out. At first, four-fold diluents were used to measure the concentrations of the conjugated forms. In second, 2-fold and 16-fold diluents were measured if glucuronyl-or sulpho-conjugates concentrations, respectively, were over the range of the calibration curve.
The urinary concentration of creatinine (Cr) was also measured using a commercial colorimetric assay (Creatinine Microplate Assay; Oxford Biomedical Research, Oxford, MI, USA). The procedures were carried out as per the manufacturer's instructions, and samples were analyzed in duplicate.

Peak specificity
Representative chromatograms of the standards of 6 conjugates of IPC, internal standards, blank urine, 4-fold diluents of mixed urine and 16-fold diluents of mixed urine are shown in Figs. 1-4. While peaks of standards and internal standards were detected as shown in Fig. 2, no peaks of these standards (6 conjugates and internal standard) were detected in blank urine at the same retention time (Fig. 1) with an exception. An adjacent peak (2.3 min) close to peak of IndG (2.5 min) was detected in 4-fold diluents of mixed urine (Fig.  3). We did not suggest the substrate derived this adjacent peak. The unknown peak at 2.3 min was not detected in blank urine (Fig. 1), therefore unknown substrate could be removed by the solid phase extraction. Hence, this unknown substrate might be closely similar molecular formula and physical property of IndG. de Loor et al. [7] did reported no adjacent peaks close to IndG in serum samples; thus, it is likely that it may be a fact observed only in urine. Nonetheless, the peak of IndG was hardly detected in 16-fold diluents of mixed urine (Fig. 4), the concentration of IndG was relatively low in human urine.
We considered our method by column separation successfully detected the conjugated forms of IPC in urine and accurately discriminated them from endogenous components. It is particularly noted that use of blank urine was excellent to detect the conjugated forms of IPC successfully, because it was suppressed matrix effects on ionization in mass spectrometry.
The results of the inter-day repeatability are shown in Table 4.  Table 5). The mean coefficient of determination for the analyzed compounds was as follows: PhG, r 2 =0.9998; IndG, r 2 =0.9969; PCG, r 2 =0.9955; PhS, r 2 =0.9996; IndS, r 2 =0.9999; PCS, r 2 =0.9984. We considered that the linearity of the calibration curve of the six conjugated forms was acceptable.

Carryover test
Carryover was not detected as peaks of standards of 6 conjugates and internal standards. The peaks did not appear in the first replicate of blank urine (data not shown).

Validation of dilutions
When compared with the peak area of blank urine, the relative peak areas of phenylacetyl-d5 L-glutamine were  Table 3. Calculated intra-day repeatability for accuracy and precision of investigated compounds.
acceptable in 2-16 fold diluents (90.7-97.1%), while we rejected in undiluted urine (80.2%; less than 85%). In addition, compared with the peak area of blank urine, the relative peak areas of p-cresyl sulfate-d7 potassium were acceptable in 4-16 fold diluents (93.2-99.5%), while we rejected in undiluted urine (67.4%) and the 2-fold diluent (82.1%). Compared with the peak area of blank urine, the relative peak areas of indoxyl-3a,4,5,6,7,7a-13 C 6 sulfate potassium were acceptable in 2-16 fold diluents (103.6-111.4%), while we rejected in undiluted urine (115.3%; more than 115%). Thus, dilution of urine was agreed 2-16 fold to analyze glucuronyl-conjugates and 4-16 fold to analyze sulpho-conjugates. The precision and concentration ratios of diluents are shown in Table 6. Precision of PhG was acceptable in 2-and 4-fold diluents (1.2 and 4.2%, respectively), while we rejected in 8-and 16-fold diluents (15.2 and 77.8%, respectively). Precision of IndG was acceptable in 2-and 4fold diluents (13.2 and 5.7%, respectively), while we rejected in 8-and 16-fold diluents (33.4 and 52.8%, respectively). Precision of PCG was acceptable in all diluents (3.8-11.6%). Precision of PhS, IndS and PCS were also acceptable in all  Our results suggested that appropriate dilution was needed to analyze the conjugated forms precisely. Four times dilution was acceptable to analyze all conjugated forms. Furthermore, consideration of some other dilution ratios was needed to include the range of calibration curve. Glucuronylconjugates were analyzed acceptable within 2 and 4-fold dilution, whereas sulpho-conjugates were analyzed acceptable within 4-16 fold dilution.

Recovery test
The recovery ratios of 3 different concentrations of the conjugated forms of IPC are shown in

Detection in clinical urine
The results of the analysis of the urinary concentrations of the conjugated forms of IPC and Cr in the six clinical urine samples are shown in Table 8. The concentrations of Glucuronyl-conjugates were lower compared with those of sulpho-conjugates. PhG was detected in two positive-type urine samples. IndG was detected only in one urine sample from a 22-year-old female donor. Although PCG was detected in five urine samples, a sample from a 32-year-old male donor was not detected. The mean concentration of PCG-positive urine was 23.5 µmol/L. PhS was detected in two urine samples from PhG-positive subjects. The mean concentration of IndS detected in all six urine samples was 143 µmol/L. PCS was detected in five urine samples, however, a sample from a 32-year-old male donor was not detected. The mean concentration of PCS in positive urine was 227 µmol/L. Poesen et al. reported that a median of PCS concentrations was 50.7 μmol/L, and a median of PCG concentration was approximately 4 μmol/L in urine of the patients with CKD [15]. These values were in range of our results (Table 8), therefore the detection methods suggesting this study seemed valuable.
Cr was detected in six clinical urine samples. All conjugated forms were detected in urine (sample from a 22year-old female) with the highest Cr concentration, but in urine (sample from a 32-year-old male) with the lowest Cr concentration only IndS was detected. The ratios of conjugated forms of IPC to Cr (IPC/Cr) are shown in Table  8. PCS/Cr and IndS/Cr were detected at high levels in all conjugated forms. Urine with the lowest concentrations of Cr Table 6. Results of the validation of dilutions. (226 mg/L) and IndS (71.1 µmol/L) had the highest IndS/Cr ratio (0.315 mmol/g) among all six urine samples. We believed that Cr correction may be important to determine the excretion of the conjugated forms of IPC in urine.

Conclusion
In the present study we demonstrated that our modified methodology of a previous procedure [7] to analyze the urinary concentration of the conjugated forms of IPC by UHPLC-MS/MS was successful. In addition, the validation of our newly-modified detection method proved the method was robust. It is worth noting that the use of blank urine was critical to detect the conjugated forms.
Although we attempted to analyze clinical urine samples by the newly-modified method, but we were unsuccessful as compounds were often undetected, even though PCS and IndS, typical toxic agents, were detected most of the time. Interestingly, the urinary concentrations of the conjugated forms of IPC seemed to depend on the concentration of Cr, suggesting that the conjugated forms of IPC and Cr should be simultaneously analyzed. For the present study, only the clinical urine samples from 6 donors were available, which were insufficient to establish a wider concentration range for each conjugate. We foresee that our modified method, which is noninvasive, will be beneficial and useful for the analysis of urine of both healthy subjects and CKD patients. We plan for the next study to improve the detection limit of the conjugates, as well as further investigation on age and sex differences and urine sampling time.  Table 8. Analysis of the concentrations of the conjugated forms of IPC and creatinine in human urine.
N.C.: not calculated. The parameters with "<" were shown without the range of calibration curve.