Should we depend on reference intervals from manufacturer package inserts? Comparing TSH and FT4 reference intervals from four manufacturers with results from modern indirect methods and the direct method.

OBJECTIVES: Correct interpretation of thyroid function tests relies on correct reference intervals (RIs) for thyroid-stimulating hormone (TSH) and free thyroxine (FT4). ISO15189 mandates periodic verification of RIs, but laboratories struggle with cost-effective approaches. We investigated whether indirect methods (utilizing historical laboratory data) could replace the direct approach (utilizing healthy reference individuals) and compared results with manufacturer-provided RIs for TSH and FT4. METHODS: We collected historical data (2008-2022) from 13 Dutch laboratories to re-establish RIs by employing indirect methods, TMC (for TSH) and refineR (for FT4). Laboratories used common automated platforms (Roche, Abbott, Beckman or Siemens). Indirect RIs (IRIs) were determined per laboratory per year and clustered per manufacturer (>1.000.000 data points per manufacturer). Direct RIs (DRIs) were established in 125 healthy individuals per platform. RESULTS: TSH IRIs remained robust over the years for all manufacturers. FT4 IRIs proved robust for three manufacturers (Roche, Beckman and Siemens), but the IRI upper reference limit (URL) of Abbott showed a decrease of 2 pmol/L from 2015. Comparison of the IRIs and DRIs for TSH and FT4 showed close agreement using adequate age-stratification. Manufacturer-provided RIs, notably Abbott, Roche and Beckman exhibited inappropriate URLs (overall difference of 0.5-1.0 µIU/mL) for TSH. For FT4, the URLs provided by Roche, Abbott and Siemens were overestimated by 1.5-3.5 pmol/L. CONCLUSIONS: These results underscore the importance of RI verification as manufacturer-provided RIs are often incorrect and RIs may not be robust. Indirect methods offer cost-effective alternatives for laboratory-specific or platform-specific verification of RIs.

Prenatal Environmental Exposure to Persistent Organic Pollutants and Reproductive Hormone Profile and Pubertal Development in Dutch Adolescents.

Persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs), may interfere with hormonal processes. Knowledge about the effects of prenatal exposure to PCBs and their hydroxylated metabolites (OH-PCBs) on pubertal development is limited. Therefore, the aim of the current study was to determine whether prenatal environmental PCB and OH-PCB exposure are associated with reproductive hormone levels and pubertal characteristics in 13- to 15-year-old children. In this Dutch observational cohort study, 194 mother-infant pairs were included (1998-2002). Maternal pregnancy serum levels of PCBs, OH-PCBs, and other POPs were measured. At follow-up (2014-2016), we measured serum or plasma levels of reproductive hormones in their children. We assessed Tanner stages and testicular volume (by clinician or standardized self-assessment), and participants completed questionnaires on pubertal onset. In total, 101 adolescents (14.4 ± 0.8 years; 53.7% of invited) participated, and 55 were boys. In boys, higher prenatal PCB levels were associated with higher testosterone levels, higher pubic hair stage, larger testicular volume, and younger age at onset of growth spurt and voice break. In girls, higher prenatal PCB levels were associated with higher stages for breast development. In conclusion, higher prenatal PCB exposure could be associated with more advanced pubertal development in 13- to 15-year-old children.

Age dependency of plasma vitamin B12 status markers in Dutch children and adolescents.

BACKGROUND: Vitamin B12 deficiency in children may be associated with (severe) neurological manifestations, therefore recognition is important. Diagnosing vitamin B12 deficiency in children is challenging. This study aimed to investigate plasma methylmalonic acid, holotranscobalamin, and total cobalamin in children 0-18 years of age and to estimate age-dependent reference intervals. METHODS: Plasma vitamin B12 markers were measured in collected plasma samples of 170 children 0-18 years visiting a local primary care laboratory. All had within-reference hemoglobin and MCV values. Pediatric plasma vitamin B12 biomarkers were measured and reference values were derived thereof. RESULTS: Plasma methylmalonic acid was higher in young children, in particular between 1 and 6 months of age; total cobalamin and holotranscobalamin were highest from 0.5 to 4 years and decreased till 10 years of age. Plasma holotranscobalamin was highly correlated with plasma total cobalamin; their ratio was independent of age. Plasma methylmalonic acid was slightly more related to total cobalamin than to holotranscobalamin. A large proportion of mainly young children would be misclassified when adult references are applied. CONCLUSIONS: Pediatric reference values for cobalamin markers are necessary to allow for early recognition and monitoring of children suspect of (clinical) cobalamin deficiency. IMPACT: We analyzed three plasma vitamin B12 status markers, i.e., total cobalamin, holotranscobalamin, and methylmalonic acid, in the plasma of 170 children 0-18 years of age and were able to derive reference intervals thereof. Recognition of vitamin B12 deficiency in children is important but challenging as pediatric reference intervals for plasma vitamin B12 status markers, particularly plasma holotranscobalamin, are not well described. We think that our results may help early recognition and monitoring of children suspect of (clinical) vitamin B12 deficiency.

In Matrix Derivatization Combined with LC-MS/MS Results in Ultrasensitive Quantification of Plasma Free Metanephrines and Catecholamines.

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated l-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 µL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4-30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (ß-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R2 > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines (R2 > 0.85). Reference intervals for l-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines.

Dermal tissue remodeling and non-osmotic sodium storage in kidney patients.

BACKGROUND: Excess dietary sodium is not only excreted by the kidneys, but can also be stored by non-osmotic binding with glycosaminoglycans in dermal connective tissue. Such storage has been associated with dermal inflammation and lymphangiogenesis. We aim to investigate if skin storage of sodium is increased in kidney patients and if this storage is associated with clinical parameters of sodium homeostasis and dermal tissue remodeling. METHODS: Abdominal skin tissue of 12 kidney patients (5 on hemodialysis) and 12 healthy kidney donors was obtained during surgery. Skin biopsies were processed for dermal sodium measurement by atomic absorption spectroscopy, and evaluated for CD68+ macrophages, CD3+ T-cells, collagen I, podoplanin + lymph vessels, and glycosaminoglycans by qRT-PCR and immunohistochemistry. RESULTS: Dermal sodium content of kidney patients did not differ from healthy individuals, but was inversely associated with plasma sodium values (p < 0.05). Compared to controls, kidney patients showed dermal tissue remodeling by increased CD68+ macrophages, CD3+ T-cells and Collagen I expression (all p < 0.05). Also, both N- and O-sulfation of heparan sulfate glycosaminoglycans were increased (all p < 0.05), most outspoken in hemodialysis patients. Plasma and urinary sodium associates with dermal lymph vessel number (both p < 0.05), whereas loss of eGFR, proteinuria and high systolic blood pressure associated with dermal macrophage density (all p < 0.05). CONCLUSION: Kidney patients did not show increased skin sodium storage compared to healthy individuals. Results do indicate that kidney failure associates with dermal inflammation, whereas increased sodium excretion and plasma sodium associate with dermal lymph vessel formation and loss of dermal sodium storage capacity. Trial registration The cohort is registered at clinicaltrials.gov as NCT (September 6, 2017). NCT, NCT03272841. Registered 6 September 2017-Retrospectively registered, https://clinicaltrials.gov.

Development and validation of a LC-MS/MS method for the establishment of reference intervals and biological variation for five plasma steroid hormones.

BACKGROUND: With liquid chromatography-tandem mass spectrometry (LC-MS/MS) increasingly being used for the quantification of steroid hormones, there is a need for studies that re-establish reference intervals and biological variation in well-defined cohorts. METHODS: A plasma steroid hormone profiling method using LC-MS/MS for quantification of progesterone, 17-hydroxyprogesterone, androstenedione, testosterone and dihydrotestosterone was developed and validated. For reference interval assessment, 280 well-characterized healthy subjects from the LifeLines cohort were selected, including 40 women using oral contraceptive pills (OCP). The biological variation was examined in 30 healthy individuals. Samples were collected over a period of 4 months with 4 week intervals. RESULTS: The developed method proved to be robust and sensitive. The reference interval levels in men are higher, whereas in women the levels tend to decrease with increasing age. In addition, women using OCP had lower levels of 17-OH-progesterone and androstenedione. The biological variation is generally higher in women compared to men, especially with regard to the inter-individual variation. CONCLUSIONS: The gender-specific determination of the reference intervals, together with the observation that the biological variation demonstrated a high degree of variation, allows interpretation of data on individual and group level for improved biochemical characterization of patients in clinical practice.

Twenty-four hour urinary cortisol excretion and the metabolic syndrome in prednisolone-treated renal transplant recipients.

Chronic prednisolone treatment in renal transplant recipients (RTR) causes metabolic abnormalities, which cluster in the metabolic syndrome (MS). It also suppresses the hypothalamic-pituitaryadrenal (HPA)-axis. We investigated whether HPA-axis suppression, as measured by 24h urinary cortisol excretion, is associated with presence of the MS and its individual components, in outpatient RTR with a functioning graft for >1year. Urinary cortisol was measured in 24h urine, using LC-MS/MS (LOQ 0.30nmol/L). We included 563 RTR (age 51±12years; 54% male) at median 6.0 [IQR, 2.6-11.5] years post-transplantation. MS was present in 439/563 RTR (78%). Median 24h urinary cortisol excretion was 2.0 [IQR, 0.9-5.1]nmol/24h. Twenty-four hour urinary cortisol excretion was independently associated with MS presence (OR=0.80 [95% CI, 0.66-0.98], P=0.02). It was also independently associated with bodyweight (st.ß=-0.11, P=0.007), waist circumference (st.ß=-0.10, P=0.01), BMI (st.ß=-0.14, P=0.001), fasting triglycerides (st.ß=-0.15, P=0.001), diabetes (st.ß=-0.12, P=0.005), and number of antihypertensives used (st.ß=-0.13, P=0.003). Suppressed HPA-axis activity, as reflected by decreased 24h urinary cortisol excretion, is associated with higher prevalence of MS and its individual components (i.e. central obesity, dyslipidemia, diabetes, hypertension) in prednisolone-treated RTR. Assessment of 24h urinary cortisol excretion by LC-MS/MS may be a tool to monitor metabolic side-effects of prednisolone in RTR.

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

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.

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-ß1 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.

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. ß-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 (ß-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 ß-blockers, diuretics and ACE inhibitors. Withdrawal of these drugs prior to the quantification of plasma metanephrines is therefore not necessary.

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

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.

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.

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.

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.

Automated mass spectrometric analysis of urinary and plasma serotonin.

Serotonin emerges as crucial neurotransmitter and hormone in a growing number of different physiologic processes. Besides extensive serotonin production previously noted in patients with metastatic carcinoid tumors, serotonin now is implicated in liver cell regeneration and bone formation. The aim was to develop a rapid, sensitive, and highly selective automated on-line solid-phase extraction method coupled to high-performance liquid chromatography-tandem mass spectrometry (XLC-MS/MS) to quantify low serotonin concentrations in matrices such as platelet-poor plasma and urine. Fifty microliters plasma or 2.5 microL urine equivalent were pre-purified by automated on-line solid-phase extraction, using weak cation exchange. Chromatography of serotonin and its deuterated internal standard was performed with hydrophilic interaction chromatography. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Serotonin concentrations were determined in platelet-poor plasma of metastatic carcinoid patients (n = 23) and healthy controls (n = 22). Urinary reference intervals were set by analyzing 24-h urine collections of 120 healthy subjects. Total run-time was 6 min. Intra- and inter-assay analytical variation were <10%. Linearity in the 0-7300 micromol/L calibration range was excellent (R(2) > 0.99). Quantification limits were 30 and 0.9 nmol/L in urine and plasma, respectively. Platelet-poor serotonin concentrations in metastatic carcinoid patients were significantly higher than in controls. The urinary reference interval was 10-78 micromol/mol creatinine. Serotonin analysis with sensitive and specific XLC-MS/MS overcomes limitations of conventional HPLC. This enables accurate quantification of serotonin for both routine diagnostic procedures and research in serotonin-related disorders.

Dietary influences on plasma and urinary metanephrines: implications for diagnosis of catecholamine-producing tumors.

CONTEXT: Measurements of the 3-O-methylated metabolites of catecholamines [metanephrines (MNs)] in plasma or urine are recommended for diagnosis of pheochromocytoma. It is unclear whether these tests are susceptible to dietary influences. OBJECTIVE: The aim of the study was to determine the short-term influence of a catecholamine-rich diet on plasma and urinary fractionated MNs. DESIGN, SETTING, AND PARTICIPANTS: We conducted a crossover study in a specialist medical center involving 26 healthy adults. INTERVENTIONS: Subjects consumed catecholamine-rich nuts and fruits at fixed times on one day (about 35 mumol dopamine and 1 mumol norepinephrine) and catecholamine-poor products on another day. Blood and urine samples were collected at timed intervals before, during, and after experimental and control interventions. MAIN OUTCOME MEASURES: Isotope-dilution mass spectrometry-based measurements of plasma and urinary concentrations of free and deconjugated 3-methoxytyramine (3-MT), normetanephrine (NMN), and MN were made. RESULTS: The catecholamine-rich diet had substantial effects (up to 3-fold increases) on plasma concentrations and urinary outputs of free and deconjugated 3-MT. Dietary catecholamines had negligible influences on free NMN in plasma and urine, but substantial effects (up to 2-fold increases) on deconjugated NMN in plasma and urine. Concentrations of free and deconjugated MN in plasma and urine remained unaffected. CONCLUSIONS: Dietary restrictions should be considered to minimize false-positive results for urinary and plasma deconjugated MNs during diagnosis of pheochromocytoma. Similar considerations appear warranted for plasma and urinary free 3-MT, but not for free NMN or MN, indicating advantages of measurements of the free compared to deconjugated metabolites.

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.

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.