Wilhelmina H. A. de Jong

Plasma tryptophan, kynurenine and 3-hydroxykynurenine measurement using automated on-line solid-phase extraction HPLC-tandem mass spectrometry

Tryptophan metabolism plays a key role in several (patho)physiological conditions. In order to study the clinical importance of tryptophan and its predominant metabolites (kynurenines), it is important to be able to measure large series of samples with high accuracy and reliability. We aimed to develop a high-throughput on-line solid-phase extraction-liquid chromatographic-tandem mass spectrometric (XLC-MS/MS) method that enables the measurement of tryptophan and its metabolites kynurenine and 3-hydroxykynurenine in plasma. Fifty microliters plasma equivalent was pre-purified by automated on-line solid-phase extraction, using strong cation exchange (PRS, propylsulphonic) cartridges. Chromatographic separation of the analytes and deuterated analogues occurred by C18 reversed phase chromatography. Mass spectrometric detection was performed in the multiple reaction-monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Total run-time including sample clean-up was 8 min. Intra- and inter-assay analytical variations were less than 9%. Linearity in the 0.11-1200 (tryptophan) and 0.050 and 0.023-45 micromol/L (kynurenine and 3-hydroxykynurenine, respectively) calibration range was excellent (R>0.99). Detection limits were 30 nmol/L for tryptophan, 1 nmol/L for kynurenine and 5 nmol/L for 3-hydroxykynurenine. Reference intervals for 120 healthy adults were 45.5-83.1 micromol/L (tryptophan), 1.14-3.02 micromol/L (kynurenine), <0.13 micromol/L (3-hydroxykynurenine) and 19.0-49.8 for tryptophan-to-kynurenine ratio. Blood sampling for tryptophan and tryptophan-to-kynurenine ratio should be performed before breakfast, due to biological variation during the day. This study describes how plasma tryptophan, kynurenine and 3-hydroxykynurenine can be measured accurately and precisely by automated high-throughput XLC-MS/MS.

Automated mass spectrometric analysis of urinary free catecholamines using on-line solid phase extraction

Analysis of catecholamines (epinephrine, norepinephrine and dopamine) in plasma and urine is used for diagnosis and treatment of catecholamine-producing tumors. Current analytical techniques for catecholamine quantification are laborious, time-consuming and technically demanding. Our aim was to develop an automated on-line solid phase extraction method coupled to high performance liquid chromatography-tandem mass spectrometry (XLC-MS/MS) for the quantification of free catecholamines in urine. Five microlitre urine equivalent was pre-purified by automated on-line solid phase extraction, using phenylboronic acid complexation. Reversed phase (pentafluorophenylpropyl column) chromatography was applied. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Urinary reference intervals were set in 24-h urine collections of 120 healthy subjects. XLC-MS/MS was compared with liquid chromatography with electrochemical detection (HPLC-ECD). Total run-time was 14 min. Intra- and inter-assay analytical variations were <10%. Linearity was excellent (R2>0.99). Quantification limits were 1.47 nmol/L, 15.8 nmol/L and 11.7 nmol/L for epinephrine, norepinephrine and dopamine, respectively. XLC-MS/MS correlated well with HPLC-ECD (correlation coefficient >0.98). Reference intervals were 1-10 micromol/mol, 10-50 micromol/mol and 60-225 micromol/mol creatinine for epinephrine, norepinephrine and dopamine, respectively. Advantages of the XLC-MS/MS catecholamine method include its high analytical performance by selective PBA affinity and high specificity and sensitivity by unique MS/MS fragmentation.

Current status and future developments of LC-MS/MS in clinical chemistry for quantification of biogenic amines

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is rapidly gaining ground in special clinical chemistry laboratories. It significantly increases the analytic potential in clinical chemistry, especially in the field of low molecular weight biomarker analysis. This review overviews current LC-MS/MS methods used for the quantification of biogenic amines and their metabolites. New possibilities offered by this technique are illustrated by recently developed assays for biogenic amines. Major shortcomings of conventional chromatographic techniques, such as labor-intensive sample preparation, long analysis times and often the relatively low specificity, are circumvented by using LC-MS/MS. In addition, LC-MS/MS has broad analyte compatibility and high analytical performance. In the last 5 years introduction of LC-MS/MS in routine diagnostics has resulted in improved assays for diagnosis and follow-up of neuroendocrine tumors characterized by the secretion of biogenic amines. Due to their labile nature and low concentration ranges biogenic amines require extensive and careful sample preparation. Introduction of new sophisticated techniques such as selective sorbents adsorption is evolving. This enables not only more specific analyte selection, but also automation of the complicated clean-up procedure. Automated sample clean-up can be directly coupled to LC-MS/MS, which facilitates reproducible and efficient handling of the growing number of samples to be analyzed in laboratories.

Urinary 5-HIAA measurement using automated on-line solid-phase extraction- high performance liquid chromatography-tandem mass spectrometry

Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful for diagnosis and follow-up of patients with carcinoid tumors and for monitoring serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method (XLC-MS/MS) that incorporates on-line solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and tandem mass spectrometric (MS/MS) detection to measure urinary 5-HIAA. Automated pre-purification of urine was carried out with HySphere-Resin GP SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard (isotope-labelled 5-HIAA-d(2)) were, after elution from the cartridge, separated by reversed-phase HPLC and detected with tandem MS. Total cycle time was 5 min. 5-HIAA and its deuterated internal standard (5-HIAA-d(2)) were retained on and eluted from the SPE cartridges in high yields (81.5-98.0%). Absolute recovery was 96.5-99.6%. Intra-assay (n=20) and inter-assay (n=20) CVs for the measurement of 5-HIAA in urine in three concentration levels ranged from 0.8 to 1.4% and 1.7 to 4.2%, respectively. For urine samples from patients (n=78) with known or suspected metastatic carcinoid tumors, results obtained by XLC-MS/MS were highly correlated (R(2)=0.99) with the routinely used fluorometric method. This XLC-MS/MS method demonstrated lower imprecision and time per analysis (high-throughput) than manual solvent extraction methods and higher sensitivity and specificity than non-mass spectrometric detection techniques.

Elevated urinary free and deconjugated catecholamines after consumption of a catecholamine-rich diet.

CONTEXT The biochemical diagnosis of pheochromocytoma depends on the demonstration of elevated levels of catecholamines (i.e. epinephrine, norepinephrine, and dopamine) and their metabolites. OBJECTIVE The aim of the study was to determine the preanalytical influence of a catecholamine-rich diet on urinary free and deconjugated catecholamines in healthy volunteers with a highly specific and sensitive analytical technique. DESIGN, SETTING, AND PARTICIPANTS We conducted a crossover study involving 27 healthy adults in a specialist medical center. INTERVENTIONS Subjects consumed catecholamine-rich nuts and fruits at fixed times on one day (about 35 micromol dopamine and 1 micromol norepinephrine) and catecholamine-poor products on another day. Urine samples were collected at timed intervals before, during, and after experimental and control interventions. MAIN OUTCOME MEASURES We performed automated online sample preparation coupled to isotope-dilution mass spectrometry measurements of urinary concentrations of free and deconjugated catecholamines. RESULTS The catecholamine-rich diet had substantial effects on urinary excretions of deconjugated dopamine (up to 20-fold increases) and norepinephrine (up to 10-fold). Dietary catecholamines had less but significant effects on urinary excretion of free dopamine and norepinephrine (up to 1.5-fold increases). Outputs of urinary free and deconjugated epinephrine remained unaffected. CONCLUSIONS Urinary excretion of deconjugated norepinephrine and dopamine is strongly affected by consumption of catecholamine-rich food products, thereby increasing the likelihood of a false-positive test result during hormonal evaluation for pheochromocytoma. Measurement of deconjugated catecholamines should therefore preferably be avoided, in favor of measurement of urinary free catecholamines. In case of demonstrating increased urinary excretion of deconjugated norepinephrine and dopamine, repeated measurements are warranted with dietary restrictions prior to sample collection.

Measurement of plasma vitamin K1 (phylloquinone) and K2 (menaquinones-4 and -7) using HPLC-tandem mass spectrometry.

Abstract Background: Given the growing interest in the health benefits of vitamin K, there is great need for development of new high-throughput methods for quantitative determination of vitamin K in plasma. We describe a simple and rapid method for measurement of plasma vitamin K1 (phylloquinone [PK]) and K2 (menaquinones [MK]-4 and -7). Furthermore, we investigated the association of fasting plasma vitamin K with functional vitamin K insufficiency in renal transplant recipients (RTR). Methods: We used HPLC-tandem mass spectrometry with atmospheric pressure chemical ionization for measurement of plasma PK, MK-4, and MK-7. Solid-phase extraction was used for sample clean-up. Mass spectrometric detection was performed in multiple reaction monitoring mode. Functional vitamin K insufficiency was defined as plasma desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) >500 pmol/L. Results: Lower limits of quantitation were 0.14 nmol/L for PK and MK-4 and 4.40 nmol/L for MK-7. Linearity up to 15 nmol/L was excellent. Mean recoveries were >92%. Fasting plasma PK concentration was associated with recent PK intake (ρ=0.41, p=0.002) and with plasma MK-4 (ρ=0.49, p<0.001). Plasma PK (ρ=0.38, p=0.003) and MK-4 (ρ=0.46, p<0.001) were strongly correlated with plasma triglyceride concentrations. Furthermore, we found that MK-4-triglyceride ratio, but not PK-triglyceride ratio, was significantly associated with functional vitamin K insufficiency (OR 0.22 [0.07–0.70], p=0.01) in RTR. Conclusions: The developed rapid and easy-to-use LC-MS/MS method for quantitative determination of PK, MK-4, and MK-7 in human plasma may be a good alternative for the labor-intensive and time-consuming LC-MS/MS methods and enables a higher sample throughput.

Unilateral and Bilateral Adrenalectomy for Pheochromocytoma Requires Adjustment of Urinary and Plasma Metanephrine Reference Ranges

CONTEXT Follow-up after adrenalectomy for pheochromocytoma is recommended because of a recurrence risk. During follow-up, plasma and/or urinary metanephrine (MN) and normetanephrine (NMN) are interpreted using reference ranges obtained in healthy subjects. OBJECTIVE Because adrenalectomy may decrease epinephrine production, we compared MN and NMN concentrations in patients after adrenalectomy to concentrations in a healthy reference population. DESIGN A single-center cohort study was performed in pheochromocytoma patients after adrenalectomy between 1980 and 2011. SUBJECTS Seventy patients after unilateral and 24 after bilateral adrenalectomy were included. MAIN OUTCOME MEASURES Plasma-free and urinary-deconjugated MN and NMN determined at 3 to 6 months and annually until 5 years after adrenalectomy were compared with concentrations in a reference population. Data are presented in median (interquartile range). RESULTS Urinary and plasma MN concentrations 3 to 6 months after unilateral adrenalectomy were lower compared with the reference population (39 [31-53] μmol/mol creatinine and 0.14 [0.09-0.18] nmol/L vs 61 [49-74] μmol/mol creatinine and 0.18 [0.13-0.23] nmol/L, respectively, both P < .05). Urinary MN after bilateral adrenalectomy was reduced even further (7 [1-22] μmol/mol creatinine; P < .05). Urinary and plasma NMN were higher after unilateral adrenalectomy (151 [117-189] μmol/mol creatinine and 0.78 [0.59-1.00] nmol/L vs 114 [98-176] μmol/mol creatinine and 0.53 [0.41-0.70] nmol/L; both P < .05). Urinary NMN after bilateral adrenalectomy was higher (177 [106-238] μmol/mol creatinine; P < .05). Changes in urinary and plasma MNs persisted during follow-up. CONCLUSION Concentrations of MN are decreased, whereas NMN concentrations are increased after unilateral and bilateral adrenalectomy. Adjusted reference values for MN and NMN are needed in the postsurgical follow-up of pheochromocytoma patients.

Determination of reference intervals for urinary steroid profiling using a newly validated GC-MS/MS method

Abstract Background: Urinary steroid profiling (USP) is a powerful diagnostic tool to asses disorders of steroidogenesis. Pre-analytical factors such as age, sex and use of oral contraceptive pills (OCP) may affect steroid hormone synthesis and metabolism. In general, USP reference intervals are not adjusted for these variables. In this study we aimed to establish such reference intervals using a newly-developed and validated gas chromatography with tandem mass spectrometry detection method (GC-MS/MS). Methods: Two hundred and forty healthy subjects aged 20–79 years, stratified into six consecutive decade groups each containing 20 males and 20 females, were included. None of the subjects used medications. In addition, 40 women aged 20–39 years using OCP were selected. A GC-MS/MS assay, using hydrolysis, solid phase extraction and double derivatization, was extensively validated and applied for determining USP reference intervals. Results: Androgen metabolite excretion declined with age in both men and women. Cortisol metabolite excretion remained constant during life in both sexes but increased in women 70–79 years of age. Progesterone metabolite excretion peaked in 30–39-year-old women and declined afterwards. Women using OCP had lower excretions of androgen metabolites, progesterone metabolites and cortisol metabolites. Method validation results met prerequisites and revealed the robustness of the GC-MS/MS method. Conclusions: We developed a new GC-MS/MS method for USP which is applicable for high throughput analysis. Widely applicable age and sex specific reference intervals for 33 metabolites and their diagnostic ratios have been defined. In addition to age and gender, USP reference intervals should be adjusted for OCP use.

No influence of antihypertensive agents on plasma free metanephrines

BACKGROUND Hypertension can be the predominant sign of pheochromocytoma (PCC) and sympathetic paraganglioma (sPGL) and screening for PCC/sPGL is often performed in patients who are already being treated with antihypertensive agents. There is very little information about the influence of antihypertensive drugs on plasma free metanephrines. The aim of this study was to determine whether commonly prescribed antihypertensive drugs can falsely elevate plasma free metanephrines concentrations measured by LC-MS/MS analysis. METHODS In a prospective study we included patients with newly diagnosed hypertension, who started monotherapy with an antihypertensive agent (i.e. β-blocker, thiazide diuretic or angiotensin-converting enzyme (ACE) inhibitor). Plasma free metanephrine (MN) and normetanephrine (NMN) levels were measured before and one month after the start of the medication quantified by LC-MS/MS. RESULTS Between 2009 and 2014, 39 patients were included (β-blocker n=13, thiazide diuretic n=14 and ACE inhibitor n=12). In the whole group, the median plasma free MN and NMN concentrations at baseline were 0.19 [0.17-0.26] nmol/L and 0.56 [0.38-0.95] nmol/L. One month after the start of antihypertensive treatment, the median plasma free MN and NMN concentrations were comparable; 0.20 [0-16-0.24] nmol/L and 0.63 [0.39-0.75] nmol/L, respectively (P=0.43 and P=0.39). Separate analysis for each of the three antihypertensive agents examined did not reveal any significant changes in the median plasma free MN and NMN concentrations. CONCLUSIONS The measurement of plasma free MN and NMN with LC-MS/MS is not affected by use of β-blockers, diuretics and ACE inhibitors. Withdrawal of these drugs prior to the quantification of plasma metanephrines is therefore not necessary.

High sodium diet converts renal proteoglycans into pro-inflammatory mediators in rats

Background High dietary sodium aggravates renal disease by affecting blood pressure and by its recently shown pro-inflammatory and pro-fibrotic effects. Moreover, pro-inflammatory modification of renal heparan sulfate (HS) can induce tissue remodeling. We aim to investigate if high sodium intake in normotensive rats converts renal HS into a pro-inflammatory phenotype, able to bind more sodium and orchestrate inflammation, fibrosis and lymphangiogenesis. Methods Wistar rats received a normal diet for 4 weeks, or 8% NaCl diet for 2 or 4 weeks. Blood pressure was monitored, and plasma, urine and tissue collected. Tissue sodium was measured by flame spectroscopy. Renal HS and tubulo-interstitial remodeling were studied by biochemical, immunohistochemical and qRT-PCR approaches. Results High sodium rats showed a transient increase in blood pressure (week 1; p<0.01) and increased sodium excretion (p<0.05) at 2 and 4 weeks compared to controls. Tubulo-interstitial T-cells, myofibroblasts and mRNA levels of VCAM1, TGF-β1 and collagen type III significantly increased after 4 weeks (all p<0.05). There was a trend for increased macrophage infiltration and lymphangiogenesis (both p = 0.07). Despite increased dermal sodium over time (p<0.05), renal concentrations remained stable. Renal HS of high sodium rats showed increased sulfation (p = 0.05), increased L-selectin binding to HS (p<0,05), and a reduction of sulfation-sensitive anti-HS mAbs JM403 (p<0.001) and 10E4 (p<0.01). Hyaluronan expression increased under high salt conditions (p<0.01) without significant changes in the chondroitin sulfate proteoglycan versican. Statistical analyses showed that sodium-induced tissue remodeling responses partly correlated with observed HS changes. Conclusion We show that high salt intake by healthy normotensive rats convert renal HS into high sulfated pro-inflammatory glycans involved in tissue remodeling events, but not in increased sodium storage.