Application of Bar Adsorptive Microextraction for the Determination of Levels of Tricyclic Antidepressants in Urine Samples.

This work entailed the development, optimization, validation, and application of a novel analytical approach, using the bar adsorptive microextraction technique (BAµE), for the determination of the six most common tricyclic antidepressants (TCAs; amitriptyline, mianserin, trimipramine, imipramine, mirtazapine and dosulepin) in urine matrices. To achieve this goal, we employed, for the first time, new generation microextraction devices coated with convenient sorbent phases, polymers and novel activated carbons prepared from biomaterial waste, in combination with large-volume-injection gas chromatography-mass spectrometry operating in selected-ion monitoring mode (LVI-GC-MS(SIM)). Preliminary assays on sorbent coatings, showed that the polymeric phases present a much more effective performance, as the tested biosorbents exhibited low efficiency for application in microextraction techniques. By using BAµE coated with C18 polymer, under optimized experimental conditions, the detection limits achieved for the six TCAs ranged from 0.2 to 1.6 µg L-1 and, weighted linear regressions resulted in remarkable linearity (r2 > 0.9960) between 10.0 and 1000.0 µg L-1. The developed analytical methodology (BAµE(C18)/LVI-GC-MS(SIM)) provided suitable matrix effects (90.2-112.9%, RSD ≤ 13.9%), high recovery yields (92.3-111.5%, RSD ≤ 12.3%) and a remarkable overall process efficiency (ranging from 84.9% to 124.3%, RSD ≤ 13.9%). The developed and validated methodology was successfully applied for screening the six TCAs in real urine matrices. The proposed analytical methodology proved to be an eco-user-friendly approach to monitor trace levels of TCAs in complex urine matrices and an outstanding analytical alternative in comparison with other microextraction-based techniques.

Osmolality-based normalization enhances statistical discrimination of untargeted metabolomic urine analysis: results from a comparative study.

INTRODUCTION: Because of its ease of collection, urine is one of the most commonly used matrices for metabolomics studies. However, unlike other biofluids, urine exhibits tremendous variability that can introduce confounding inconsistency during result interpretation. Despite many existing techniques to normalize urine samples, there is still no consensus on either which method is most appropriate or how to evaluate these methods. OBJECTIVES: To investigate the impact of several methods and combinations of methods conventionally used in urine metabolomics on the statistical discrimination of two groups in a simple metabolomics study. METHODS: We applied 14 different strategies of normalization to forty urine samples analysed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). To evaluate the impact of these different strategies, we relied on the ability of each method to reduce confounding variability while retaining variability of interest, as well as the predictability of statistical models. RESULTS: Among all tested normalization methods, osmolality-based normalization gave the best results. Moreover, we demonstrated that normalization using a specific dilution prior to the analysis outperformed post-acquisition normalization. We also demonstrated that the combination of various normalization methods does not necessarily improve statistical discrimination. CONCLUSIONS: This study re-emphasized the importance of normalizing urine samples for metabolomics studies. In addition, it appeared that the choice of method had a significant impact on result quality. Consequently, we suggest osmolality-based normalization as the best method for normalizing urine samples. TRIAL REGISTRATION NUMBER: NCT03335644.

The Urinary Polyomavirus-Haufen Test: A Highly Predictive Non-Invasive Biomarker to Distinguish "Presumptive" from "Definitive" Polyomavirus Nephropathy: How to Use It-When to Use It-How Does It Compare to PCR Based Assays?

"Definitive" biopsy proven polyomavirus nephropathy (PyVN), usually caused by BK polyomavirus (BKPyV), remains a significant infection of kidney transplants. Diagnosis depends upon an allograft biopsy and outcome depends upon early intervention. Here, we report data on a non-invasive biomarker for PyVN, the urinary PyV-Haufen test. Test results were compared to those of conventional laboratory assays targeting PyV replication, i.e., BKPy-viremia, -viruria and urinary decoy cell shedding. Of 809 kidney transplant recipients, 228 (28%) showed PyV replication with decoy cell shedding and/or BKPy-viremia by quantitative PCR; only a subset of 81/228 (36%) showed "definitive" PyVN. Sensitivity and specificity for identifying patients with PyVN was: 100% and 98%, respectively, urinary PyV-Haufen test; 50% and 54%, respectively, urinary decoy cell shedding; 97% and 32%, respectively, BKPy-viremia with cut-off of ≥250 viral copies/mL; 66% and 80%, respectively, for BKPy-viremia ≥104 viral copies/mL. The PyV-Haufen test showed a very strong correlation with the severity of PyVN (Spearman's ρ = 0.84) and the Banff PyVN disease classes (p < 0.001). In comparison, BKPy-viremia and -viruria levels by PCR displayed modest correlations with PyVN severity (Spearman's ρ = 0.35 and 0.36, respectively) and were not significantly associated with disease classes. No association was found between decoy cell shedding and PyVN severity or disease classes. Pilot data demonstrated that PyVN resolution with decreasing Banff pvl-scores was reflected by a gradual decrease in PyV-Haufen shedding; such a tight association was not noted for BKPy-viremia. In conclusion, urinary PyV-Haufen testing is a highly specific, non-invasive method to accurately diagnose patients with "definitive" PyVN and to optimize patient management. Assay specifics are discussed.

A new 7-point method facilitates accurate diagnosis of high-grade urothelial carcinoma in routine urinary cytology practice.

INTRODUCTION: This study was designed to identify the minimal and necessary cell morphologies to be considered for high-precision diagnosis of high-grade urothelial carcinoma (HGUC) in a routine urinary cytology practice. MATERIALS AND METHODS: We included 338 urine cytology specimens from 11 medical facilities in Japan. Six experts evaluated these Papanicolaou-stained specimens using their own diagnostic criteria to categorize them within an initial 4-tiered classification system. Of the 338 cases, 70 HGUC and 32 benign cases (with a complete consensus diagnosis of 6 experts) were included for the analysis. Two of the cytologists evaluated the specimens for 20 specific cellular features. The results were analyzed using a contingency table and by discriminant analysis. RESULTS: Of the original 338 cases, 165 were originally diagnosed as HGUC, but only 70 (42.4%) were scored as malignant by all participating cytologists; of the 101 benign cases, only 32 (31.7%) were classified as such in all examinations. These specimens were re-evaluated by 6 experts using a panel of 20 specific cellular features used to distinguish between HGUC and benign diseases; tests of significance and discriminant analyses identified 7 critical features that were most useful for cytological diagnosis. Statistical analysis revealed that a focus on these 7 features led to a diagnosis of HGUC with a probability of over 95%. CONCLUSIONS: The accuracy of our presently used method to evaluate urinary cytology is not consistently high. This novel classification system, which focuses on 7 critical features, facilitates the high accurate diagnosis of HGUC in routine cytology practice.

Evaluation of urinary inflammatory index in rapid screening of urinary tract infection.

The objective of this study was to assess the diagnosis value of urinary inflammatory index (UII) and systemic immune-inflammation index (SII) for UTI. Nine inflammatory indexes including neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, SII and six UIIs were calculated for Receiver operating characteristic curve analysis to select which one is suitable for the screening of UTIs or distinguishing the types of bacteria. UII3, which calculated from leucocyte esterase (LE), nitrite, white blood cells and bacteria, was preferentially used as an indicator for the diagnosis of UTI when the threshold was set at 0.53. UII2 was more suitable for the distinction between groups when the cutoff is set to 0.94. Appropriate urinary inflammation index calculated by rapid urinalysis of urine dipstick and urine sediment can help us to predict urinary tract infection and bacterial type, and reduce the workload and costs of urine culture.

Assessment of Interobserver Reliability of Nephrologist Examination of Urine Sediment.

Importance: Urine sediment microscopy is commonly performed during the evaluation of kidney disease. Interobserver reliability of nephrologists' urine sediment examination has not been well studied. Objective: Assess interobserver reliability of the urine sediment examination. Design, Setting, and Participants: In this diagnostic test study, urine samples were prospectively collected from a convenience sample of adult patients from an academic hospital in the United States undergoing kidney biopsy from July 11, 2018, to March 20, 2019. Digital images and videos of urine sediment findings were captured using a bright-field microscope. These images and videos along with urine dipstick results were incorporated in online surveys and sent to expert nephrologists at 15 US teaching hospitals. They were asked to identify individual sediment findings and the most likely underlying disease process. Exposures: Urine dipstick results and urine sediment images from patients undergoing native kidney biopsy. Main Outcomes and Measures: Interobserver reliability of urine sediment microscopy findings estimated by overall percent agreement and Fleiss κ coefficients. Secondary outcomes included concordance of diagnoses suspected by nephrologists with corresponding kidney biopsy results. Results: In total, 10 surveys from 10 patients containing 76 study questions on individual features were sent to 21 nephrologists, 14 (67%) of whom completed them all. Their combined 1064 responses were analyzed. Overall percent agreement for casts was an estimated 59% (95% CI, 50%-69%), κ = 0.52 (95% CI, 0.42-0.62). For other sediment findings, overall percent agreement was an estimated 69% (95% CI, 61%-77%), κ = 0.65 (95% CI, 0.56-0.73). The κ estimates ranged from 0.13 (95% CI, 0.10-0.17) for mixed cellular casts to 0.90 (95% CI, 0.87-0.94) for squamous epithelial cells. Conclusions and Relevance: In this study, substantial variability occurred in the interpretation of urine sediment findings, even among expert nephrologists. Educational or technological innovations may help improve the urine sediment as a diagnostic tool.

Using urine to diagnose large-scale mtDNA deletions in adult patients.

OBJECTIVE: The aim of this study was to evaluate if urinary sediment cells offered a robust alternative to muscle biopsy for the diagnosis of single mtDNA deletions. METHODS: Eleven adult patients with progressive external ophthalmoplegia and a known single mtDNA deletion were investigated. Urinary sediment cells were used to isolate DNA, which was then subjected to long-range polymerase chain reaction. Where available, the patient`s muscle DNA was studied in parallel. Breakpoint and thus deletion size were identified using both Sanger sequencing and next generation sequencing. The level of heteroplasmy was determined using quantitative polymerase chain reaction. RESULTS: We identified the deletion in urine in 9 of 11 cases giving a sensitivity of 80%. Breakpoints and deletion size were readily detectable in DNA extracted from urine. Mean heteroplasmy level in urine was 38% ± 26 (range 8 - 84%), and 57% ± 28 (range 12 - 94%) in muscle. While the heteroplasmy level in urinary sediment cells differed from that in muscle, we did find a statistically significant correlation between these two levels (R = 0.714, P = 0.031(Pearson correlation)). INTERPRETATION: Our findings suggest that urine can be used to screen patients suspected clinically of having a single mtDNA deletion. Based on our data, the use of urine could considerably reduce the need for muscle biopsy in this patient group.

The art of obtaining a high yield of cell-free DNA from urine.

Although liquid biopsies offer many advantages over tissue biopsies, they are not yet standard practice. An important reason for the lack of implementation is the unavailability of well standardized techniques and guidelines, especially for pre-analytical conditions which are an important factor causing the current sensitivity issues. To overcome these limitations, we investigated the effect of several pre-analytical conditions on the concentration of cell-free DNA (cfDNA) and cellular genomic DNA (gDNA) contamination. Urine samples from healthy volunteers (HVs) and cancer patients were collected and processed according to specific pre-analytical conditions. Our results show that in samples with a relatively small volume more than 50% of the cfDNA can be found in the first 50 mL of the urine sample. The total DNA concentration increased again when samples were collected more than 3.5 hours apart. Adding preservative to urine samples is recommended to obtain high concentrations of cfDNA. To remove the cellular content, high speed centrifugation protocols as 4,000g 10min or 3,000g 15min are ideal for urine collected in cfDNA Urine Preserve (Streck). Although this study was a pilot study and needs to be confirmed in a larger study population, clear trends in the effect of several pre-analytical conditions were observed.

Reference intervals of spot urine copper excretion in preschool children and potential application in pre-symptomatic screening of Wilson disease.

The objectives were to determine the reference intervals of spot urine copper excretion indexes in pre-school children and to evaluate their utility in screening for Wilson disease (WD). With spot urine collected from a control sample of preschool children (aged 3-7 years, n=153), the reference intervals of spot urine copper excretion indexes and their biological variation were defined. In order to investigate their utility performance in screening for WD in this age group, multiple spot urine samples from six WD patients who were diagnosed at presymptomatic stage were also analysed and compared. Cut-off values useful for detection of WD were defined by receiver operator curve (ROC) analysis. Biological (inter-individual) variation of spot urine copper indexes expressed as coefficient of variation (CVg) were around 60% at this age group, which was moderate and similar to other clinically useful urine tests, such as urine albumin excretion ratio. Spot urine copper excretion strongly correlated with both urine creatinine and osmolality. Linear regression against both creatinine and osmolality showed that ∼94% of data points in healthy preschool children fell within the prediction interval, suggesting that both were useful normalisation factors. ROC showed that copper to osmolality ratio was the best index with an area under curve (AUC) greater than 0.98. Cut-off values of 0.5 µmol/L, 0.1 µmol/mmol and 0.00085 µmol/mOsmol (32 µg/L, 56 µg/g creatinine and 0.054 µg/mOsmol, respectively, in conventional units) for spot urine copper concentration, copper to creatinine ratio and copper to osmolality ratio, respectively, have potential application in the differentiation of WD patients. Based on the data, a new WD screening strategy targeting preschool children is proposed. Application of a bivariate screening strategy using spot urine copper concentration and urine osmolality may be useful in a population-wide screening program for WD among preschool children.

Update on urine as a biomarker in cancer: a necessary review of an old story.

Introduction: Cancer causes thousands of deaths worldwide each year. Therefore, monitoring of health status and the early diagnosis of cancer using noninvasive assays, such as the analysis of molecular biomarkers in urine, is essential. However, effective biomarkers for early diagnosis of cancer have not been established in many types of cancer.Areas covered: In this review, we discuss recent findings with regard to the use of urine composition as a biomarker in eleven types of cancer. We also highlight the use of urine biomarkers for improving early diagnosis.Expert opinion: Urinary biomarkers have been applied for clinical application of early diagnosis. The main limitation is a lack of integrated approaches for identification of new biomarkers in most cancer. The utilization of urinary biomarker detection will be promoted by improved detection methods and new data from different types of cancers. With the development of precision medicine, urinary biomarkers will play an increasingly important clinical role. Future early diagnosis would benefit from changes in the utilization of urinary biomarkers.

Pheochromocytoma: An approach to diagnosis.

Pheochromocytomas are rare neuroendocrine chromaffin-derived tumors that arise within the adrenal medulla. They are usually benign, but if not diagnosed or if left untreated, they can have devastating consequences. Clinical consideration of the diagnosis is paramount, as they may have protean manifestations, and a high index of suspicion is essential if serious consequences are to be avoided. An accurate biochemical diagnosis is crucial for the management of these patients: either plasma or urinary metanephrines are both highly sensitive and specific if correctly employed, but knowledge of pre- and post-analytic interference is essential. Diagnostic imaging with cross-sectional CT and/or MRI offers high sensitivity in their detection, but lack specificity. The introduction of PET/CT/MR has led to a dramatic improvement in the localization of both pheochromocytomas and paragangliomas, together with the increasing availability of new functional imaging radionuclides. Optimal investigation and accurate diagnosis is best achieved at 'centers of excellence' with expert multidisciplinary teams.

Current and emerging bladder cancer biomarkers with an emphasis on urine biomarkers.

Introduction: Bladder cancer detection typically requires unpleasant and costly cystoscopy, a procedure potentially harmful and often accompanied by variable adverse effects. The use of urine analysis as a noninvasive method is of great scientific interest since it is enriched in tumor-related proteins, DNA and RNA which can provide a molecular landscape with multiple alterations identified in bladder cancer.Areas covered: Current sensitivity, specificity and diagnostic accuracy of FDA approved urine-based assays are still suboptimal with none of them routinely used by clinics. The recent introduction of RNA/DNA based bladder cancer tests, some of them commercially available, establishes a promising new horizon of clinical applicability.Expert opinion: There is growing evidence toward the use of minimally invasive 'liquid biopsies' to identify biomarkers in urothelial malignancy. Urine has been identified as an optimal noninvasive source of proteins, DNA and RNA; therefore, it has been identified as a type of liquid biopsy likely to soon be routine clinical practice. Cell-free proteins and peptides, exosomes, cell-free DNA, methylated DNA and DNA mutations, circulating tumor cells, miRNA, lncRNA, rtRNA and mRNAs, have been assessed in urine specimens. However, lack of well-designed multicenter clinical studies remain as important limitation, and therefore, precludes their use in clinical practice.

Screening for Microscopic Hematuria in a Urogynecologic Population.

OBJECTIVES: The objectives of this were to determine the correlation of greater than or equal to 3 red blood cells per high-power field (RBCs/HPF) with a positive urine dipstick for blood and to identify clinically relevant factors than can influence this relationship. METHODS: The charts of women with positive blood urine dipsticks were reviewed from August 2012 to August 2013. The cohort of women was divided into 2 groups; those with urine with greater than or equal to 3 RBCs/HPF on microscopy and those without. Relevant clinical and demographic variables were extracted from the electronic medical record. Data analysis was conducted using SAS version 9.4 (SAS Institute, Cary, NC). RESULTS: Most of the 203 patients eligible for analysis were Caucasian, and the total cohort had a mean age of approximately 62.8 years. Microscopy confirmed greater than or equal to 3 RBCs/HPF in 25.6% of the urine samples. A dipstick finding of moderate or large blood was significantly more likely to have greater than or equal to 3 RBCs/HPF on univariate and multivariable analyses (P < 0.001). Factors significantly associated with greater than or equal to 3 RBCs/HPF were increasing age, recurrent urinary tract infections, and urinary specific gravity of greater than 1.010. CONCLUSIONS: Lower urinary specific gravities appear to be associated with underestimating microhematuria, likely owing to the underrepresentation of the true number of red blood cells. Urine dipstick indicators of moderate or large blood increase the likelihood the microscopy samples demonstrated greater than or equal to 3 RBCs/HPF. These findings suggest that clarification of microhematuria detection and evaluation guidelines should be considered, given both important clinical and economic consequences.

Risk of Contamination of Voided Urine Specimen in Women With Pelvic Organ Prolapse.

OBJECTIVES: The primary aim of this study was to determine if urine cultures are more likely to be contaminated in women with pelvic organ prolapse (POP). The secondary aim was to evaluate the test characteristics of a urine dipstick in women with POP. METHODS: A retrospective cohort study was conducted of women who presented to the urogynecology clinic between September 1, 2017, and August 31, 2018. Associations between the presence of POP and contaminated urine culture results were estimated using univariable and multivariable analyses. The sensitivity and specificity of a urine dipstick in women with POP were calculated. RESULTS: We included 351 women (143 with and 208 without POP). Women with POP were older (65.4 ± 15.8 vs 60.7 ± 11.0 years, P < 0.01), had a lower body mass index (26.6 ± 4.8 vs 29.2 ± 7.7 kg/m, P < 0.01), and were less likely to have recurrent urinary tract infections (3.5% vs 9.6%, P=0.03). Women with POP were more likely to have a contaminated urine culture than women without POP (55.9% vs 40.9%, P < 0.01). Rates of contaminated urine culture were higher in women with stage 3 and 4 prolapse than in women with stage 2 prolapse (59.6% vs 41.0%, P < 0.01). On multivariate analysis, the odds of contaminated urine culture remained higher in women with POP (odds ratio, 1.89; 95% confidence interval, 1.20-2.99). In women with POP, the sensitivity (23.5%) and positive predictive value (66.7%) of a urine dipstick were poor. CONCLUSIONS: Women with POP are more likely to provide a contaminated urine culture when collecting a midstream urine specimen.

Utility of DNA Next-Generation Sequencing and Expanded Quantitative Urine Culture in Diagnosis and Management of Chronic or Persistent Lower Urinary Tract Symptoms.

Many patients suffer from chronic, irritative lower urinary tract symptoms (LUTS). The evaluation and management of these patients have proven difficult with the use of standard diagnostic tools, including urinalysis and urine culture. The growing body of literature on the urinary microbiome has looked at the possible implications of the bladder microbiome and dysbiosis, or perturbations in the microbiome, in conditions associated with chronic LUTS. Disorders such as recurrent urinary tract infections (UTIs) and interstitial cystitis have been studied utilizing 16S rRNA rapid next-generation gene sequencing (NGS) and expanded quantitative urine culture (EQUC). In this article, we first present a brief review of the literature describing the current understanding of the urinary microbiome and the features and applications of NGS and EQUC. Next, we discuss the conditions most commonly associated with chronic, persistent LUTS and present the limitations of current diagnostic practices utilized in this patient population. We then review the limited data available surrounding treatment efficacy and clinical outcomes in patients who have been managed based on results provided by these two recently established diagnostic tools (DNA NGS and/or EQUC). Finally, we propose a variety of clinical scenarios in which the use of these two techniques may affect patients' clinical outcomes.

Effect of changing urine testing orderables and clinician order sets on inpatient urine culture testing: Analysis from a large academic medical center.

OBJECTIVE: To evaluate the impact of changes to urine testing orderables in computerized physician order entry (CPOE) system on urine culturing practices. DESIGN: Retrospective before-and-after study. SETTING: A 1,250-bed academic tertiary-care referral center. PATIENTS: Hospitalized adults who had ≥1 urine culture performed during their stay. INTERVENTION: The intervention (implemented in April 2017) consisted of notifications to providers, changes to order sets, and inclusion of the new urine culture reflex tests in commonly used order sets. We compared the urine culture rates before the intervention (January 2015 to April 2016) and after the intervention (May 2016 to August 2017), adjusting for temporal trends. RESULTS: During the study period, 18,954 inpatients (median age, 62 years; 68.8% white and 52.3% female) had 24,569 urine cultures ordered. Overall, 6,662 urine cultures (27%) were positive. The urine culturing rate decreased significantly in the postintervention period for any specimen type (38.1 per 1,000 patient days preintervention vs 20.9 per 1,000 patient days postintervention; P < .001), clean catch (30.0 vs 18.7; P < .001) and catheterized urine (7.8 vs 1.9; P < .001). Using an interrupted time series model, urine culture rates decreased for all specimen types (P < .05). CONCLUSIONS: Our intervention of changes to order sets and inclusion of the new urine culture reflex tests resulted in a 45% reduction in the urine cultures ordered. CPOE system format plays a vital role in reducing the burden of unnecessary urine cultures and should be implemented in combination with other efforts.

Performance of the point-of-care circulating cathodic antigen (POC-CCA) urine cassette test for follow-up after treatment of S. mansoni infection in Eritrean refugees.

BACKGROUND: Point-of-care circulating cathodic antigen (POC-CCA) urine cassette testing has become a popular approach to screen for Schistosoma infection. Since the test is also increasingly used for following-up of treatment success, we assessed the assay's diagnostic accuracy after praziquantel treatment of S. mansoni infection among Eritrean refugees in Switzerland. METHODS: In our preceding study, 107 asymptomatic Eritrean refugees in Switzerland were screened for schistosomiasis by stool microscopy, serology, and POC-CCA urine testing. Individuals screened positive by any method were treated with praziquantel and invited for a follow-up visit, repeating the same diagnostic procedures one year after treatment. The POC-CCA baseline and follow-up results were analyzed against the 'baseline microscopy positive cases' (= the most reliably true positive cases) and the 'baseline microscopy plus serology negative cases at baseline and follow-up' (= the most reliably true negative cases). RESULTS: Complete diagnostic baseline and follow-up sampling was available from 48 participants. Compared to most reliably true positive cases at baseline, POC-CCA testing had a sensitivity of 90%. Compared to most reliably true negative cases, POC-CCA testing had a specificity of 73.9%. CONCLUSION: We conclude that the POC-CCA urine test is valuable for screening but its use is not suitable for routine follow-up after treatment.

Use of diagnostic stewardship practices to improve urine culturing among SHEA Research Network hospitals.

This survey investigated interventions used by acute-care hospitals to reduce the detection of asymptomatic bacteriuria. Half of the respondents reported using reflex urine cultures but with varied urinalysis criteria and perceived outcomes. Other diagnostic stewardship interventions for urine culture ordering and specimen quality were less common.

Urinary strips for proteins: easy to do but difficult to read!

Urine is an easily accessible biological fluid. This availability makes it tempting for a direct analysis of urinary parameters by physicians. Urinary proteins, that are interesting markers for several pathologies, are detected by urinary strips, rapidly and at a low cost. However, to ensure an optimal use of urinary strips, one may be familiar to their characteristics and their limits. This paper details the available urinary strips used for the detection of urinary proteins and urinary albumin, as well as their analytical performances in clinical conditions such as pregnancy, chronic renal disease, diabetes or for the screening of general populations.

A critical evaluation of the use of gas chromatography- and high performance liquid chromatography-mass spectrometry techniques for the analysis of microbial metabolites in human urine after consumption of orange juice.

The present study compared and validated two analytical methods, HPLC-HRMS, and GC-MS using MSTFA as derivatization agent, for the analysis of microbiota-derived phenolic acids and aromatic compounds accumulating in urine, collected over a 24 h period after the consumption of 500 mL of orange juice. In addition, purification procedures using SDB-L and HLB solid phase cartridges were compared when HPLC-HRMS technique was used. Both HPLC-HRMS and GC-MS methodologies were successfully validated in terms of specificity, sensitivity, limit of detection and quantification, recovery and matrix effects. HPLC-HRMS, unlike GC-MS, does not require sample derivatization prior to analysis. GC-MS was not suitable for the analysis of phenolic sulfate and glucuronide metabolites because of their lack of volatility. These phase II metabolites could, however, be analysed by HPLC-HRMS which, as a consequence, provided more detailed and complete information on the phenolic compounds derived from microbiota-mediated degradation of orange juice (poly)phenols. Furthermore, the use of SDB-L and HLB cartridges for sample purification prior to HPLC-HRMS analysis is suitable for free phenolics and glucuronide metabolites but not sulfate derivatives. These findings highlight that the use of an inappropriate analytical protocol can adversely affect studies on the bioavailability of dietary (poly)phenols in which microbiota-derived phenolic catabolites play an important role.