Our goal was to develop and validate a targeted high-throughput UPLC-TQ-MS/MS method for simultaneous detection and quantification of phenylacetylglutamine, 4-cresyl sulphate and hippurate, and to quantify concentrations of these gut microbial co-metabolites in 4,000 human urine specimens obtained from the INTERMAP Study. We used a Waters ACQUITY™
UPLC system coupled with a Waters Xevo™
triple quadrupole mass spectrometer (Waters Corporation, Manchester, UK). LC-MS was selected over other analytical platforms such as LC-UV and NMR, since it is a more appropriate, faster and convenient analytical platform for targeted quantitative analysis21
Development of the targeted UPLC-MS/MS method
An initial method development process showed that the selected metabolites required detection in different ionization modes: phenylacetylglutamine was detected in positive electrospray ionization (ESI) mode (), while 4-cresyl sulphate and hippurate were detected in negative ESI mode (). Hippurate was also detected in positive ESI mode, but potential for interferences were apparent, so for this analysis negative mode acquisition was used. Consequently each urine specimen was injected twice in order to accommodate detection in both ESI modes.
Fig. 1 UPLC/MS MRM chromatograms showing the retention times of the three human urinary microbial co-metabolites multiple transitions. (Figure 1A) Analytes and 2H internal standards detected in ESI(+) mode, (Figure 1B) analytes and 2H internal standards detected (more ...)
For detection of each analyte and deuterated internal standard, multiple reaction monitoring (MRM) transitions of a precursor ion [M+H]+ (for molecules ionizing in positive ion mode) or [M−H]− (for molecules ionizing in negative ion mode) into a characteristic fragment ion were recorded. The MRM transitions used for integration and quantification were: m/z 265>129 for phenylacetylglutamine, m/z 270>129 for 2H5-phenylacetylglutamine in positive ESI mode, and m/z 187>106 for 4-cresyl sulphate, m/z 190>110 for 2H3-4-cresyl sulphate, m/z 178>76 for hippurate, m/z 180>76 for 2H2-hippurate in negative ESI mode. ESI parameters, selection of MS/MS transitions, and conditions were established by direct infusion of each standard, followed by optimization utilizing in-built automated Waters software (IntelliStart™). Using the reported MRM transitions, no significant endogenous interfering peaks were observed in the blank wells for the analytes or the internal standards, indicating that there was no carry over or contamination.
The initial phase of the study also involved the optimization of analytical conditions. This involved measuring 1mg/mL stock solutions of each reference standard using the UPLC-MS/MS system, to achieve a short gradient and separation time. As a result, the total experimental time necessary to acquire the full set of concentration data was 5 minutes per urine specimen (including two injections per specimen run in both positive and negative ESI modes). Parameters such as gas flow rates, temperature and capillary voltages were investigated for each individual metabolite. However, the only parameters specific to each metabolite were cone voltage and the collision energy ().
Table 1 UPLC-MS/MS conditions and retention times for the three analysed urinary metabolites and associated labelled standards. Separations were performed under isocratic conditions (95:5) of water plus 0.1% formic acid:acetonitrile plus 0.1% formic acid. A mobile (more ...)
We validated linearity using calibrators at concentrations 1, 3, 10, 30, 100, 300 and 1000ng/mL for phenylacetylglutamine and 4-cresyl sulphate, and 3, 10, 30, 100, 300, 1000 and 3000ng/mL for hippurate made up in water. It was not possible to make up the standard curves by spiking into the analytical matrix (urine), since the natural urinary abundances of these endogenous metabolites were high (especially hippurate). Thus a very high concentration of internal standard would have had to be spiked into the matrix, which would have saturated the source. Calibration curves were established for each analyte based on the ratio of integrated area under the peak of each calibrator divided by that of the spiked-in deuterated internal standard used to quantify each metabolite in each specimen. We found that for the best fit of the calibration curve a weighted (1/x) linear regression gave R2 > 0.997 for phenylacetylglutamine and 4-cresyl sulphate, whereas R2 > 0.997 was obtained for hippurate by fitting to a weighted (1/x2) second order regression (calculated by Waters TargetLynx™ 4.1 software). Acceptance criteria were <15% deviation of calibrators from nominal concentrations (<20% at the LLOQ).
Additional experiments were carried out to validate the developed method according to published US Food and Drug Administration (FDA) guidelines22
. To evaluate imprecision and recovery (inaccuracy), we analyzed multi-analyte QC samples at three concentration levels: low (3ng/ml for phenylacetylglutamine and 4-cresyl sulphate, 10ng/mL for hippurate), medium (30ng/ml for phenylacetylglutamine and 4-cresyl sulphate, 100ng/mL for hippurate) and high (300ng/ml for phenylacetylglutamine and 4-cresyl sulphate, 1000ng/mL for hippurate) using six replicates on three separate well plates. Imprecision (CV) was calculated as the standard deviation divided by the mean of the detected concentration and expressed as a percentage. Recovery (inaccuracy) was calculated as the agreement between measured analyte concentration and nominal (theoretical) target concentration. Intra-assay results (differences in measured concentration between the replicates on the same plate) and inter-assay results (differences in measured concentration between replicates on 3 different plates) were determined and are given in . The CVs determined at three concentrations were within ±15% of the theoretical values, except for 4-cresyl sulphate at 3ng/mL for the inter-assay assessment, which had a CV estimate of 16.7%. Thus, precision and recovery of the method were mostly acceptable according to FDA guidelines22
Intra and inter-assay imprecision and recovery data of the three analytes, calculated for the validation of the UPLC-MS/MS method developed in this study.
As the assay used MS/MS detection, it was necessary to assess the likelihood of matrix effects23,24
. A matrix effect is the suppression or enhancement of ionization of analytes by the presence of matrix components in biological samples. This can be especially detrimental in a quantitative assay, since a matrix effect may suppress or enhance analyte response, leading to underestimation or overestimation of the true concentration measurement24
. Therefore, to check for the presence of matrix effects and to determine the selectivity and reliability of our quantification method, we performed a parallelism of dilution experiment, where analyte concentrations were measured following serial dilution of the matrix (human urine specimens). The results, given in Supplemental Data Table 1
show that after multiplying each measured concentration by its respective dilution factor, the concentrations were almost identical (with the exception of one, anomalous result for hippurate at a dilution of x200). These data confirmed that there were no interferences from other endogenous urinary metabolites, indicating the absence of matrix effects and demonstrating specificity and reliability.
Application of the developed method to large scale population samples
Using the UPLC-MS/MS method described here, we analyzed 4,000 urine specimens from 2,000 US INTERMAP participants (two specimens/person) in 500 hours. The targeted method developed here reliably measured three urinary metabolites at concentrations as low as 1ng/mL (phenylacetylglutamine and 4-cresyl sulphate) and 3ng/mL (hippurate). In order to obtain absolute quantification, metabolite concentrations were analyzed via addition of deuterated isotope internal standards. Stable isotope internal standards are an ideal choice for LC-MS quantification assays25
, as their physicochemical properties are similar to those of the analyte. Therefore, in addition to co-eluting and undergoing the same ionization conditions, they also provide control over matrix effects26
. Analytical specificity of the method was confirmed by the ability to differentiate and quantify the metabolites of interest in the presence of many other compounds in the urinary specimens.
While potential sources of error have been reported previously for UPLC-MS/MS quantification methods such as ionization inaccuracy, non-specificity, imprecision and inaccuracy relating to instrument and handling (e.g. sample preparation)27, 28
, we took steps to mitigate these potential limitations in our study. This included using the most appropriate isotope labeled internal standards (deuterated and non-deuterated 4-cresol suphate was custom synthesised in-house), performing a method validation study prior to analyzing the INTERMAP urine specimens to ensure there were no matrix effects, and including QC samples on each analytical plate to monitor imprecision and inaccuracy. Supplemental Data Table 2
gives method validation data following analysis of the 4,000 urine specimens: mean concentration, % deviation of the mean concentration from nominal concentrations, imprecision and recovery (inaccuracy) of the six QC samples included in each batch. The quantification data from the QC samples distributed throughout the 50 batch run demonstrate that the method and instrument were robust and reproducible over multiple batches. Precision and recovery values for the QC samples did not decline over the duration of the study, attributable to the analysis of 13L injections of predominantly aqueous urine specimens. Sample preparation and chromatographic separation times were minimized, making the assay suitable for high-throughput applications, such as large-scale epidemiologic and biobank studies.
Reference ranges for Phenylacetylglutamine, 4-Cresyl Sulphate and Hippurate in Human Urine
Our analysis provides reference ranges for urinary concentrations and 24-hr urinary output of phenylacetylglutamine, 4-cresyl sulphate and hippurate for free-living multi-ethnic US men and women aged 40–59 (). Mean concentrations, (standard deviation) based on the mean of two specimens/person were: for urinary phenylacetylglutamine, 81.0 (48.9) μmol/mmol creatinine and 1283.0 (751.7) μmol/24-hr (men), 113.9 (64.3) μmol/mmol creatinine and 1145.9 (635.5) μmol/24-hr (women); for 4-cresyl sulphate, 63.0 (47.4) μmol/mmol creatinine and 1002.5 (737.1) μmol/24-hr (men), 103.1 (71.2) μmol/mmol creatinine and 1031.8 (687.9) μmol/24-hr (women); for hippurate, 398.0 (265.4) μmol/mmol creatinine and 6284.6 (4008.1) μmol/24-hr (men), 476.8 (340.1) μmol/mmol creatinine and 4793.0 (3293.3) μmol/24-hr (women).
Reference ranges for human urinary concentrations of phenylacetylglutamine (PAG), 4-cresyl sulphate (4CS) and hippurate, given in ng/mL, μmol/mmol creatinine (Cr.), and μmol/24-hr.
Phenylacetylglutamine and 4-cresyl sulphate are derived from biotransformation of tryptophan and tyrosine respectively, whereas hippurate is the glycine conjugate of benzoic acid which is introduced via a range of plant and other dietary sources. Mean urinary excretion of phenylacetylglutamine was previously found to be 1080.0 μmol/24-hr in seven normal adults (gender unspecified)29
via isotope dilution GC-MS. Our results are consistent with these previous data, as we observed mean excretion of 1283.0 μmol/24-hr in men and 1145.9 μmol/24-hr in women. To our knowledge the present study is the first to report a reference range for human urinary 4-cresyl sulphate excretion, and so provides a unique resource for clinical chemistry measurements.
Mean urinary hippurate concentrations were previously reported to be 175.9 (SD 124.3) μmol/mmol creatinine in men and 207.3 (SD 118.8) μmol/mmol creatinine in women in a Greek population study30
compared with 398.0 μmol/mmol creatinine and 476.8 μmol/mmol creatinine respectively in our study. Since hippurate precursors are present in plant and other dietary sources31
, this difference may be attributable to dietary differences between the Greek and multi-ethnic US population samples in our study. Furthermore, the previous study was based on a smaller sample than our own (N=122 vs. N=2000). Our newly generated reference ranges shown in , indicate that total 24-hr urinary excretion of phenylacetylglutamine and 4-cresyl sulphate are similar in men and women, whereas 24-hr hippurate excretion is considerably higher in men compared to women. Extant literature reported higher hippurate excretion in women compared with men30
, based on μmol/mmol creatinine concentrations. We also observed this μmol/mmol creatinine concentration gender difference; however, since our study included replicate measurements (second 24-hr urine collection) on each individual, we are able to report 24-hr urinary excretion values. These may be more informative than concentration data since 24-hr urinary excretion measurements take into account diurnal variation and intra-individual differences in creatinine excretion relating to body mass and the responsiveness to meat in the diet. Our results also show an age related trend with higher 24-hr excretion of all three microbial metabolites, at ages 50–59 compared with 40–49 years (, ).
Fig. 2 Box and whisker plots for phenylacetylglutamine, 4-cresyl sulphate, and hippurate (μmol/24-h), by gender, age and ethnic group. Corresponding statistics are given in and Supplemental Data Table 3. Diamond=mean; central horizontal line=median; (more ...)
The 2,000 INTERMAP US individuals in this study are from several ethnic groups. When urinary excretion differences were compared between ethnic groups (, Supplemental Data Table 3
), we found that urinary excretion of all three metabolites were significantly (p
<0.05) lower in the Japanese subpopulation compared with other ethnic groups; most Japanese participants were from Hawaii with perhaps a more traditional lifestyle than on mainland USA. 24-hr phenylacetylglutamine and 4-cresyl sulphate excretion were not significantly different between White Non Hispanic, African American, Hispanic Non White and Hispanic White ethnic groups. For 24-hr urinary hippurate excretion White Non Hispanic participants excreted significantly higher amounts of hippurate compared with the other subpopulations (twice the 24-hr excretion of the Japanese subgroup). African American, Hispanic Non White and Hispanic White had similar 24-hr hippurate excretions.