Urinary metanephrines by liquid chromatography tandem mass spectrometry: Using multiple quantification methods to minimize interferences in a high throughput method

Journal of Chromatography B, 879 (2011) 3673–3680

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Urinary metanephrines by liquid chromatography tandem mass spectrometry: Using multiple quantification methods to minimize interferences in a high throughput method夽 Zlatuse D. Clark a , Elizabeth L. Frank b,∗ a b

ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112, United States

a r t i c l e

i n f o

Article history: Received 14 May 2011 Accepted 3 October 2011 Available online 11 October 2011 Keywords: Liquid chromatography Mass spectrometry Metanephrine Normetanephrine Pheochromocytoma Solid phase extraction

a b s t r a c t Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC–MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC–MS method. Injection-to-injection time was 6 min. The validated LC–MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Pheochromocytoma, a neuroendocrine tumor arising from the chromaffin cells of the adrenal medulla, is a rare and often overlooked cause of secondary hypertension [1]. Although most pheochromocytomas are benign, tumor production of excess catecholamines has potentially fatal consequences due to the potent impact of these compounds on the cardiovascular system [2].

Abbreviations: ESI, electrospray ionization; GC–MS, gas chromatography mass spectrometry; LC–MS/MS, liquid chromatography tandem mass spectrometry; LOD, limit of detection; LOQ, limit of quantitation; MN, metanephrine; MRM, multiple reaction monitoring; NMN, normetanephrine; QIR, qualitative ion ratio; SPE, solid phase extraction. 夽 Parts of these data were presented at the MSACL conference in San Diego, CA, February 2010, and at the annual meeting of the American Association for Clinical Chemistry in Anaheim, CA, July 2010. ∗ Corresponding author. Tel.: +1 801 583 2787x2087. E-mail address: [email protected] (E.L. Frank). 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.10.005

Therefore, correct diagnosis and timely treatment of these tumors are crucial. Over the last several decades, various biochemical tests for catecholamines and their metabolites have been used for differential diagnosis [3]. The measurement of plasma free and/or urinary fractionated metanephrines was recommended as appropriate testing for initial assessment by the First International Symposium on Pheochromocytoma [4]. While plasma metanephrines have been reported to have a higher clinical diagnostic value than urinary metanephrines [5–7], both tests have significant advantages over tests for the parent catecholamines and other metabolites [4]. Assessment of urine metanephrine excretion has been proposed as the appropriate test for screening a population in which the pre-test probability of disease is low [8]. Some of the advantages of testing for urinary as opposed to plasma metanephrines are higher analyte concentrations, which makes analysis less challenging, and a noninvasive sample collection that minimizes expenditure of time and effort by the medical staff [4]. In our laboratory, urinary metanephrines is a high-volume test frequently requested by medical providers. Previously, this

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test was performed using a GC–MS method [9]. Although a significant improvement over lower throughput HPLC assays with high incidence of interferences, the GC–MS methods suffer from longer run times and more cumbersome sample preparation than required for an LC–MS/MS platform. The increasing availability of this powerful technique in clinical laboratories is evidenced by the growing number of publications describing LC–MS/MS methods for urinary [10–12] and plasma metanephrines [13–15]. This study describes the development and validation of a simple, reliable, high throughput LC–MS/MS method for the analysis of metanephrine and normetanephrine in urine. Potential interferences are mitigated by the use of multiple quantification methods.

phosphate buffer, pH 7 (0.2 mol/L), and 30 ␮L of 6 mol/L NaOH were added to neutralize samples. Solid phase extraction (SPE) was performed using a 96-well positive pressure extraction manifold (SPEware Corp., Baldwin Park, CA). Samples were vortexed briefly and applied onto a polymeric weak cation exchanger (PWCX) 96well SPE plate (SPEware) conditioned with 1 mL of methanol and equilibrated with 1 mL of water. Each well was washed with equal volumes (1.8 mL) of water and 70% methanol. Analytes were eluted in 500 ␮L of 2% formic acid in 5% methanol. The eluate was injected directly onto the LC column.

2. Experimental

Instrumental analysis was performed on an AB Sciex API 3200 triple quadrupole mass spectrometer, with TurboIonSpray interface, interfaced with CTC Pal autosampler and an Agilent 1200 LC system with a degasser and a column oven. Data were acquired in multiple reaction monitoring (MRM) mode. Instrument control and data analysis were performed using AB Sciex Analyst® software, version 1.4.2. Chromatographic separation was achieved using an Ultra II PFP propyl column (2.1 mm × 50 mm, 3-␮m particles) with an integrated pre-filter assembly (Restek, Bellefonte, PA). The compounds were eluted isocratically using 0.2% formic acid in 5% methanol followed by a gradient to 50% methanol to clean the column. The mobile phase flow rate was 500 ␮L/min, the column temperature was 30 ◦ C, and the injection volume was 15 ␮L. Electrospray ionization was performed in positive mode. Purified nitrogen was used for the nebulizing (517 kPa, 75 psi), auxiliary (517 kPa, 75 psi), curtain (172 kPa, 25 psi) and collision gases (34.5 kPa, 5 psi). The interface heater temperature was 600 ◦ C. The TurboIonSpray capillary voltage was set to 1500 V. The declustering potential was 42 V for MN and its internal standard transitions and 33 V for NMN and its internal standard transitions. The entrance potential was 11 V and the collision energies were 21–25 V. MRM transitions were monitored at m/z: 180 → 165 (quantifier I), 180 → 148 (quantifier II), and 180 → 120 (qualifier) for MN; 183 → 168 (quantifier I), 183 → 151 (quantifier II) and 183 → 123 (qualifier) for MN-d3 ; 166 → 134 (quantifier) and 166 → 106 (qualifier) for NMN; and 169 → 137 (quantifier) and 169 → 109 (qualifier) for NMN-d3 , with 100 ms dwell time for each transition. A qualitative ion ratio (QIR) was used to indicate the presence of interferences. The QIRs were calculated as ratio of analyte concentration for the quantifier transition to analyte concentration for the qualifier transition. Patient specimen QIR acceptability range was established as ±30% of the mean value of the QIRs obtained from the calibration standards.

2.1. Chemicals and reagents HPLC-grade methanol was purchased from JT-Baker; formic acid (99%) and hydrochloric acid (HCl, 37%) from VWR (West Chester, PA); and sodium hydroxide (NaOH), sodium hydrogen phosphate and sodium dihydrogen phosphate from Sigma–Aldrich (St. Louis, MO). Racemic metanephrine·HCl was purchased from Toronto Research Chemicals (North York, Ontario, CAN); racnormetanephrine·HCl, dopamine·HCl, 3-methoxytyramine·HCl, rac-norepinephrine (+)-bitartrate, and rac-epinephrine base from Sigma–Aldrich; rac-metanephrine-d3 ·HCl (␣1, ␤2) from Cambridge Isotope Laboratories, Inc. (Andover, MA); rac-normetanephrined3 ·HCl (␣1, ␤2) was from Medical Isotopes, Inc. (Pelham, NH). Lyphochek Quantitative Urine Controls I and II were purchased from BioRad Clinical Division (Hercules, CA). NANOpure water, obtained from a Barnstead water system, was used throughout the study. 2.2. Standards and specimens All individual catecholamine and metanephrine standard stock solutions were prepared in 0.2% aqueous formic acid at 0.5 mmol/L. For direct infusion experiments, the stock solutions were diluted to 2.5 ␮mol/L with 0.2% formic acid in 50% methanol. For the signal suppression experiments, the metanephrine and normetanephrine stock solutions were diluted to 10 ␮mol/L in water. Metanephrine-d3 (MN-d3 ) and normetanephrine-d3 (NMN-d3 ) combined working internal standard solution was prepared in 5 mmol/L aqueous HCl at 12.5 ␮mol/L. Calibration and linearity standards were prepared at 25, 100, 500, 2000, 7000 and 10,000 nmol/L concentrations by dilution of the 0.5 mmol/L combined metanephrine (MN) and normetanephrine (NMN) stock solution in 5 mmol/L aqueous HCl. Control materials were prepared at three concentrations by reconstitution of Lyphochek Quantitative Urine Controls in 50 mmol/L aqueous HCl. Two control concentrations were chosen to approximate the medical decision limits (upper limit of reference interval) for the analytes. Concentrations for the third control were set at the low end of the calibration curve. Aliquots of stock solutions were stored frozen at −70 ◦ C. Urine specimens for testing were selected from the routine workload, de-identified and handled according to guidelines approved by the Institutional Review Board of the University of Utah (IRB #7275). 2.3. Sample preparation Working internal standard solution (50 ␮L) was added to 250 ␮L of urine specimen, controls and calibration standards. Samples were acidified with 30 ␮L of 6 mol/L HCl, incubated at 90 ◦ C in an aluminum dry heat block (VWR) for 15 min, and cooled during centrifugation (2000 g) at 5 ◦ C for 5 min. One milliliter of sodium

2.4. LC–MS/MS instrumentation and conditions

2.5. Method validation 2.5.1. Linearity Linearity was evaluated by analyzing extracted combined MN and NMN standards at 25, 100, 500, 2000, 7000 and 10,000 nmol/L. Each standard was analyzed once in 20 different runs on 20 d. The acceptance criterion for linearity was ±10% of expected concentration. 2.5.2. Analytical sensitivity Sensitivity was evaluated for both the quantitative and qualitative transitions by analyzing extracted combined MN and NMN standards prepared at 2.5, 5, 10, 25, 50 and 100 nmol/L concentrations. Each standard was analyzed 10 times. The limit of quantification (LOQ) was defined as the lowest concentration for which the CV was within 20% and the concentration was within 20% of the expected value. The limit of detection (LOD) was defined as the lowest concentration at which the analyte peak was present

Z.D. Clark, E.L. Frank / J. Chromatogr. B 879 (2011) 3673–3680

in both the quantitative and the qualitative mass transitions at the expected retention time and the signal to noise ratio for the quantitative mass transition was greater than 5.

3675

NE

3.0e5

E

2.5.4. Method comparison The newly developed LC–MS/MS method was compared to the GC–MS method in use. Specimens were stored at or below −20 ◦ C prior to analysis. 2.5.5. Recovery Recovery was evaluated using pooled human urine with low MN and NMN concentrations, 63 and 151 nmol/L, respectively, spiked with the analyte standards at 50, 500, 1500, and 5000 nmol/L. All measurements were carried out in triplicate and the mean values were used for calculations. Four types of recovery parameters were calculated for each analyte concentration: extraction efficiency (RE) as analyte peak area in urine spiked pre-extraction divided by analyte peak area in urine spiked post-extraction; matrix effect (ME) as analyte peak area in urine spiked post-extraction divided by analyte peak area in clean solvent; process efficiency (PE) as analyte peak area in urine spiked pre-extraction divided by analyte peak area in clean solvent; and internal standard normalized recovery (NR) as analyte concentration in urine spiked pre-extraction divided by analyte concentration in clean solvent. The peak areas/concentrations obtained for the pooled urine matrix were subtracted from the peak areas/concentrations measured for the spiked sample aliquots [16]. 2.5.6. Evaluation of ion suppression Ion suppression was evaluated as the presence of negative peaks at the retention time of MN and NMN. Extracted patient samples were injected onto the LC column and individual 10 ␮mol/L analyte solutions were co-infused post-column as described by Annesley [17]. 2.5.7. Analytical specificity Epinephrine, norepinephrine, dopamine and 3methoxytyramine were evaluated with MN and NMN during method development. The chromatographic conditions were chosen such that all of these compounds showed baseline separation. Complete separation was especially important for epinephrine and normetanephrine, as these two compounds are isobaric and, therefore, epinephrine has the potential to interfere with normetanephrine quantification. In a separate experiment, additional endogenous compounds structurally similar to metanephrines as well as a number of drugs, such as analgesics and anti-hypertensives, were tested for interference with the assay. The potential interferences were spiked into the low control at two concentrations (5 and 20 mg/L) before extraction. In addition to the MRM mode, the baseline sample and the spiked samples were also analyzed in the Q1MS mode to determine the retention times and primary ions for the potentially interfering compounds. 2.5.8. Carryover Carryover was evaluated at 50,000 nmol/L as described in EP Evaluator software. A 25 nmol/L combined MN and NMN standard was used for a low concentration specimen; a 50,000 nmol/L standard was used as the high concentration specimen.

Intensity (cps)

3-MT

2.5.3. Imprecision Method imprecision was evaluated by analyzing control materials at three concentrations over 20 d in two replicates per run, one run per day.

NMN D

2.0e5

MN

1.0e5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Time (min) Fig. 1. LC–MS/MS separation of catecholamine and metanephrine standards: norepinephrine (NE), epinephrine (E), normetanephrine (NMN), dopamine (D), metanephrine (MN), and 3-methoxytyramine (3-MT).

2.5.9. Reference interval Previously established reference intervals were verified by analyzing 127 de-identified 24-h urine specimens submitted for routine testing for trace metals. The analyte concentrations were recalculated for total urine volume and values in nmol/d were used for reference interval verification. 2.5.10. Data analysis Statistical evaluation of the data was performed according to CLSI guidelines using EP Evaluator software, release 8 (David G. Rhoads Associates, Inc.). 3. Results 3.1. Sample preparation and LC–MS/MS conditions The acid hydrolysis procedure used in our previous GC–MS method [9] was re-optimized. Several combinations of final HCl concentration (0.3, 0.6, and 3 M), temperature (20, 60 and 90 ◦ C), and duration (15 and 30 min) were evaluated using standard solutions (50 and 4000 nmol/L), three quality control materials as well as low (90 nmol/L for MN and 250 nmol/L for NM) and high (5300 nmol/L for both MN and NMN) urine pools. Measurements were carried out in duplicate. Analyte concentration yields were determined for each combination of conditions. The highest yields for all samples tested were obtained with 0.6 M final HCl concentration at 90 ◦ C for either 15 or 30 min. Since there was no statistical difference between analyte yields at 15 and 30 min, the shorter time was used in the re-optimized procedure. For sample cleanup, several types of SPE sorbents as well as combinations of wash and elution solvents were tested. The use of polymeric mixed mode cation exchange sorbent and an eluent compatible with the initial LC mobile phase conditions allowed for direct injection of the extract onto the LC column. Atlantis T3 (Waters); Luna HILIC, Kinetex HILIC and Kinetex PFP (Phenomenex); Allure PFP propyl and Ultra II PFP propyl (Restek) LC columns were tested. The Ultra II PFP propyl column provided the best chromatographic separation of MN, NMN, 3methoxytyramine and the closely related catecholamines. Optimal mobile phase conditions were 0.2% formic acid and 5% methanol during initial isocratic separation of the analytes, with a gradient to 50% methanol to clean the column (Fig. 1). HILIC columns did not yield sufficient separation of the compounds under any conditions tested.

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MS parameters were determined using direct infusion and flow injection analysis of 2.5 ␮mol/L analyte solutions in 0.2% formic acid in 50% methanol. As shown previously (10), the protonated molecular ions of metanephrines readily undergo loss of water in the ESI source, yielding the more stable [M−H2 O+1]+ ions, which were selected as the precursor ions. MRM transitions utilizing the loss of water were avoided because of their lack of specificity. Typical chromatograms of metanephrine standards and of healthy and abnormal patient specimens are shown in Fig. 2.

a

MN-d3

5.0e5

4.0e5

Intensity (cps)

3676

NMN-d3 3.0e5

2.0e5

MN 1.0e5

3.2. Method validation results 3.2.1. Linearity The assay was linear to 10,000 nmol/L. Linearity data are shown in Table 1. Observed concentrations for MN and NMN were plotted against expected concentrations to give Eqs. (1) and (2), respectively.

Normetanephrine :

observed error 1.6%. (2)

Specimens with analyte concentrations exceeding the upper limit of linearity were diluted up to 25-fold.

1.0

1.5

b

2.0

2.5

3.0

3.5

4.0

3.0

3.5

4.0

3.0

3.5

4.0

MN-d3

4.0e5

observed error 2.7%. (1)

y = 0.996x + 0.42;

0.5

Time (min)

Intensity (cps)

y = 1.002x − 0.96;

Metanephrine :

0.0

NMN

3.2.2. Analytical sensitivity The LOD was 2.5 nmol/L for both analytes. The LOQ requirements were met at 10 nmol/L for both MN transitions and for the NMN qualifier transition, and at 5 nmol/L for NMN quantifier transition (Table 2). The method LOQ was accepted as 10 nmol/L.

3.0e5

NMN-d3

2.0e5

1.0e5

0.0

NMN

0.5

MN

1.0

1.5

2.0

2.5

Time (min)

3.2.4. Method comparison Specimens (n = 286) with analyte concentrations spanning the linearity range were assayed in the method comparison experiment (Fig. 3). Data above the upper limit of linearity and those with QIRs outside of the ±30% limits were excluded. Good agreement was observed between the GC–MS method and the newly developed LC–MS/MS method for both MN and NMN. Deming regression equations for MN, Eq. (3), and NMN, Eq. (4), along with standard errors and correlation coefficients were: y = 1.050x − 45.4;

Metanephrine : R = 0.989;

Sy/x = 105.0;

n = 275 y = 1.062x − 158.8;

c

5.0e5

Sy/x = 421.4;

MN-d3

3.0e5

MN 2.0e5

NMN-d3

1.0e5

0.0

(3)

NMN

4.0e5

Intensity (cps)

3.2.3. Imprecision Within-run, between-day, and total imprecision for all three quality control concentrations were between 1.0 and 2.7% (Table 3).

0.5

1.0

1.5

2.0

2.5

Time (min)

(4)

Fig. 2. LC–MS/MS chromatograms of (a) 200 nmol/L calibrator, (b) healthy patient urine, and (c) abnormal patient urine: normetanephrine (NMN), d3 normetanephrine (d3 -NMN), metanephrine (MN), d3 -metanephrine (d3 -MN).

3.2.5. Recovery Numerous forms of recovery calculations, with varying degrees of clarity on how the calculations were performed, have been published in the literature. In this study, we conducted a comprehensive recovery experiment and calculated four recovery-related parameters that are pertinent to LC–MS/MS methods: extraction efficiency (RE), quantifying analyte losses during sample preparation; matrix effect (ME), indicating ionization suppression (ME < 100%) or enhancement (ME > 100%); process efficiency (PE), measuring the net effect of extraction efficiency and matrix effect on the final response of the analyte [16]; and recovery normalized

to IS (NR), calculated as a ratio of analyte concentration to that of its IS. The values for each analyte and concentration along with mean values ± SD are given in Table 4. While for MN the higher RE (90 ± 4%) and the lower signal suppression (ME = 88 ± 3%) produced higher PE (79 ± 2%) compared to NMN (RE = 78 ± 3%, ME = 83 ± 6%, PE = 65 ± 7%), the high NRs (MN: 99 ± 2%, NMN: 97 ± 5%) showed that the isotopically labeled internal standards compensated for extraction losses as well as the relatively small matrix effects for both analytes.

Normetanephrine : R = 0.970;

n = 273

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Table 1 Linearity data for the determination of urinary metanephrines by LC–MS/MS. Standard concentration (nmol/L)

25 100 500 2000 7000 10000

Metanephrine

Normetanephrine

Mean (nmol/L)

Recovery (%)

Precision CV (%)

Mean (nmol/L)

Recovery (%)

Precision CV (%)

23.5 102 513 2057 6930 9757

93.8 102 103 103 99.0 97.6

2.7 2.3 1.7 1.3 0.4 1.3

24.9 99.8 497 2026 6975 9803

99.7 99.8 99.3 101 99.6 98.0

3.0 1.7 2.3 1.5 0.4 1.7

Table 2 Sensitivity data for the determination of urinary metanephrines by LC–MS/MS. Expected concentration (nmol/L)

Mean meas. concentration (nmol/L)

Fitted CV (%)

Metanephrine (quantifier I) 1.5 38.5 3.6 16.3 8.2 7.6 3.1 24.0 49.9 1.8 1.3 101.3 Metanephrine (qualifier/quantifier II) 3.0 54.4 4.3 38.0 8.8 17.3 5.0 23.8 50.0 1.2 100.6 −0.6

2.5 5 10 25 50 100 2.5 5 10 25 50 100

Accuracy (%)

Mean meas. concentration (nmol/L) Normetanephrine (quantifier) 3.0 4.7 9.1 23.9 49.5 98.6 Normetanephrine (qualifier) 3.5 5.6 10.2 25.4 49.9 100.4

60 72 82 96 100 101 121 85 88 95 100 101

Fitted CV (%)

Accuracy (%)

21.4 14.1 7.8 3.6 2.3 1.7

119 94 91 95 99 99

32.5 20.3 10.9 4.0 1.8 0.6

142 112 102 101 100 100

Table 3 Imprecision data for the determination of urinary metanephrines by LC–MS/MS. Metanephrine

Normetanephrine

Mean (nmol/L)

Within-run CV (%)

Between-day CV (%)

Total CV (%)

Mean (nmol/L)

Within-run CV (%)

Between-day CV (%)

Total CV (%)

123.7 945.9 1817.7

1.6 1.0 1.6

0.9 2.5 1.3

1.9 2.7 2.0

431.8 3148.6 4724.6

1.9 1.5 1.2

1.1 2.1 2.2

2.2 2.6 2.5

Table 4 Recovery data for the determination of urinary metanephrines by LC–MS/MS. Concentration of added analyte (nmol/L)

Metanephrine a

50 500 1500 5000 Mean (SD) a b c d

Normetanephrine b

c

d

RE (%)

ME (%)

PE (%)

NR (%)

REa (%)

MEb (%)

PEc (%)

NRd (%)

96 87 89 89 90 (4)

84 88 88 91 88 (3)

81 76 78 81 79 (2)

99 99 100 96 99 (2)

76 75 80 82 78 (3)

75 84 84 90 83 (6)

53 63 67 73 64 (8)

90 102 98 99 97 (5)

RE: extraction efficiency. ME: matrix effect. PE: process efficiency. NR: internal standard normalized recovery.

3.2.6. Evaluation of ion suppression The co-infusion test showed no negative peaks at the retention times of the analytes confirming that there was no appreciable signal suppression from co-elution of interfering substances. However, we observed signal suppression of approximately 10% for MN and 20% for NMN caused by incomplete signal recovery after the elution of low molecular weight ionic species in the solvent front. The deuterated internal standards compensated well for the signal suppression observed.

3.2.7. Analytical specificity Compounds evaluated as potential interferences are listed in Table 5; MN and NMN data are shown for comparison. Percent bias, calculated as a ratio of analyte concentrations in the

spiked vs. baseline sample, was used as a measure of interference. The bias for all tested compounds was 6% or less for both analytes indicating no interference from any of the compounds tested. The samples were analyzed also in Q1MS mode under the same LC conditions to identify the retention times and primary ions of the compounds tested as potential interferences (Table 5). 3.2.8. Carryover Both MN and NMN passed the carryover test at 50,000 nmol/L; that is, the calculated carryover values (1.36 and 3.12 nmol/L, respectively) were less than the error limits (1.95 and 3.54 nmol/L, respectively), defined as three times the SD of two consecutive low results.

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Table 5 Retention times, molecular weights, and primary observed ions for analytes and potential interferences. Potential interference/analyte

Retention time (min)

MW (g/mol)

Norepinephrine Epinephrine Normetanephrine Dopamine l-Tyrosine Nicotine Metanephrine Carbidopa Isoproterenol 3,4-Methylenedioxyamphetamine 3-Methoxytyramine Hydralazine Serotonin Cimetidine Phenylpropanolamine Isoetharine Levodopa Caffeine Ephedrine Pseudoephedrine Amphetamine Vanillylmandelic acid Homovanillic acid 5-Hydroxyindoleacetic acid Phenoxybenzamine Labetalol Theophylline Theobromine Acetaminophen Acetylsalicylic acid Fenfluramine Amitriptyline Cocaine Methamphetamine 3,4-Methylenedioxymethamphetamine Diazepam

0.55 0.77 0.95 1.13 1.27 1.41 1.70 2.23 2.49 2.50 2.62 2.63 2.75 2.77 2.83 2.99 3.05 3.13 3.25 3.34 3.89 Not found Not found Not found Not found Not found Not found Not found Not found Not found Not found Not found Not found Not found Not found Not found

169 183 183 153 181 162 197 226 211 179 167 160 176 252 151 239 197 194 165 165 135 198 183 191 304 328 180 180 151 180 231 277 303 149 183 285

3.2.9. Reference interval The reference intervals established for our GC–MS method (152–1775 nmol/d for MN, 273–3550 nmol/d for NMN) were verified. The observed ranges (n = 127) were: for MN, 96–1751 nmol/d; 1.7% results outside (max 10%), and for NMN 409–4533 nmol/d; 3.9% results outside (max 10%). 3.3. Method improvements After the original method implementation, several improvements were incorporated to further optimize method performance. To reduce the number of specimens requiring dilution, the upper limit of linearity was increased to 15,000 nmol/L (validation data not shown). In the newly developed method, the m/z 180 → 165 and 180 → 148 were originally chosen as the MN quantifier and qualifier transitions, respectively, to provide the highest analytical sensitivity. However, routine use revealed that the incidence of unknown interferences was approximately 3–4%, not the anticipated 2% calculated from the validation data. To mitigate the interference problems, we added third MRM transitions for both MN (m/z 180 → 120) and MN-d3 (m/z 183 → 123) to the acquisition method. No interference was observed for either of these transitions during a validation study of 1976 patient specimens. Two separate quantification methods were programmed: a primary method, using the m/z 180 → 165 (quantifier I) and m/z 180 → 120 (qualifier) transitions, and an alternate method, using the m/z 180 → 148 (quantifier II) and m/z 180 → 120 (qualifier) transitions for MN. Acquired data were processed with the primary quantification method. Data for samples with MN QIRs

Primary ion (m/z) 152.2 166.2 166.2 137.2 182.1 163.1 180.2 227.1 212.3 180.1 151.2 161.2 177.2 254.1 152.3 240.1 177.2 195.2 166.2 166.2 136.3 – – – – – – – – – – – – – – –

outside of the 30% acceptance limits were reprocessed with the alternate quantification method. Implementation of the two quantification methods decreased the overall incidence of nonreportable results from 3.7% to 0.15%. The very small percentage of non-reportable results was due to the fact that a few specimens displayed significant concurrent interferences on both the m/z 180 → 165 and the m/z 180 → 148 transitions and their QIRs failed using both the primary and the alternate quantification methods.

4. Discussion In current practice urinary and/or plasma metanephrines are the biochemical tests of choice for the diagnosis and assessment of pheochromocytoma. Some argue that while the clinical sensitivity of both tests is similar, plasma metanephrines supersede urinary metanephrines in clinical specificity [5,6]. However, the concentrations of metanephrines in plasma are 2–3 orders of magnitude lower than those in urine, presenting a greater analytical challenge. Consequently, urinary metanephrine testing is frequently requested. A GC–MS method developed in our laboratory [9] had been reliably accommodating our urinary metanephrine test volume for almost a decade. The method employed a double derivatization step that increased its sensitivity and specificity, but made the sample preparation cumbersome and time consuming. Furthermore, it became increasingly difficult to obtain acceptable quality and quantity of the derivatizing reagents, and the decision was made to transfer the assay to an LC–MS/MS platform.

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a

7000

Metanephrine

LC-MS/MS (nmol/L)

6000 5000 4000 3000 2000 1000 0 0

1000 2000 3000 4000 5000 6000 7000

GC-MS (nmol/L)

b 12000

Normetanephrine

LC-MS/MS (nmol/L)

10000 8000 6000 4000 2000

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but also decreased the incidence of other interferences. Since urinary catecholamines are a frequently requested test, concurrent analysis of catecholamines and metanephrines seems appealing. However, as indicated by other authors [11,12], catecholamines usually are analyzed in their free form, whereas reference intervals for metanephrines are based on measuring total compound concentrations after acid hydrolysis. Consequently, the two groups of analytes require separate sample preparation, and combining them is not advantageous at the present. The third metanephrine, 3-methoxytyramine, on the other hand, can easily be included in the newly developed assay. Although not offered as part of this assay, the analyte does have a clinical diagnostic value. Van Duinen et al. [18] have shown that urinary 3-methoxytyramine can be used to identify dopamine-producing paragangliomas and is a more sensitive marker than urinary dopamine. We intend to add 3-MT to our assay in the near future. For metanephrines, selection of sensitive transitions free of interferences is a challenge due to their small size and similarity to other endogenous compounds as well as pharmaceuticals. Despite our thorough investigation of potential interferences, significant unidentified interferences on the MN m/z 180 → 165 and 180 → 148 transitions occurred in approximately 3–4% of patient specimens. Therefore we added a third transition (m/z 180 → 120), for which interferences were not observed, to the acquisition method and programmed primary and alternate quantification methods. In a study of 1976 patient specimens, this approach decreased the overall incidence of non-reportable results from 3.7% to 0.15%, while preserving throughput. It is important to note that in our analysis of over 2000 specimens, all of the samples with failed MN QIRs had MN excretion below the upper limit of the reference interval. Thus, the observed interferences did not affect the method’s ability to detect patients with pheochromocytoma.

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GC-MS (nmol/L) Fig. 3. Comparison of results obtained using the LC–MS/MS method with the established GC–MS method for (a) MN and (b) NMN.

The improvement goals set to tailor the new method to the needs of the laboratory included: reduced specimen volume, shorter sample preparation time, higher analytical throughput, and a potential for automation. In the newly developed LC–MS/MS method the volume of urine was reduced from 500 ␮L to 250 ␮L. The acid hydrolysis procedure was shortened from 30 min to 15 min. The use of a cation exchange sorbent and an eluent compatible with the LC mobile phase allowed for direct injection of the eluate onto the LC column, thus eliminating an evaporation step. Also, MN and NMN ionize easily using electrospray ionization technique; therefore, derivatization is not necessary. The injectionto-injection analysis time was shortened from 13 min to 6 min, doubling the throughput. Consequently, an assay that previously required two GC–MS instruments is now performed on a single LC–MS/MS system. Individual SPE cartridges were replaced with a 96-well platform amenable to automation. Automation of sample preparation using a liquid handler and the use of column switching to further increase method throughput are additional improvements to be implemented. Although not included in the final method, epinephrine, norepinephrine, dopamine and 3-methoxytyramine were evaluated with MN and NMN during the chromatographic optimization. LC conditions were chosen to achieve baseline separation of all compounds. This not only eliminated the potential for interference for the isobaric compounds epinephrine and normetanephrine,

5. Conclusions In conclusion, we have developed, thoroughly characterized and validated a robust, high-throughput method for the determination of urinary metanephrines. This method utilizes a simple LC–MS/MS system and a 96-well format SPE amenable to automation. Occasional interferences occurring on the two MN transitions chosen initially were resolved by addition of a third transition and the use of two quantification methods. The assay was placed into service in August 2010 and has performed exceptionally well. Acknowledgements We would like to thank Dr. Mark Kushnir, Mr. Seyed Sadjadi and the staff of the Mass Spectrometry 2 Laboratory for their assistance during this project. This study was supported by the ARUP Institute for Clinical and Experimental Pathology® . References [1] J.W. Lenders, G. Eisenhofer, M. Mannelli, K. Pacak, Lancet 366 (2005) 665. [2] W.M. Manger, R.W. Gifford, J. Clin. Hypertens. (Greenwich) 4 (2002) 62. [3] T.G. Rosano, G. Eisenhofer, R.J. Whitley, Catecholamines and serotonin, in: C. Burtis, E. Ashwood, D. Bruns (Eds.), Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed., Elsevier Saunders, St. Louis, 2006, p. 1033. [4] K. Pacak, G. Eisenhofer, H. Ahlman, S.R. Bornstein, A.P. Gimenez-Roqueplo, A.B. Grossman, N. Kimura, M. Mannelli, A.M. McNicol, A.S. Tischler, Nat. Clin. Pract. Endocrinol. Metab. 3 (2007) 92. [5] J.W. Lenders, K. Pacak, M.M. Walther, W.M. Linehan, M. Mannelli, P. Friberg, H.R. Keiser, D.S. Goldstein, G. Eisenhofer, J. Am. Med. Assoc. 287 (2002) 1427. [6] N. Unger, C. Pitt, I.L. Schmidt, M.K. Walz, K.W. Schmid, T. Philipp, K. Mann, S. Petersenn, Eur. J. Endocrinol. 154 (2006) 409. [7] G. Eisenhofer, I.J. Kopin, D.S. Goldstein, Pharmacol. Rev. 56 (2004) 331. [8] K.L. Brain, J. Kay, B. Shine, Clin. Chem. 52 (2006) 2060. [9] D.K. Crockett, E.L. Frank, W.L. Roberts, Clin. Chem. 48 (2002) 332.

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[10] R.L. Taylor, R.J. Singh, Clin. Chem. 48 (2002) 533. [11] M.J. Whiting, Ann. Clin. Biochem. 46 (2009) 129. [12] R. Marrington, J. Johnston, S. Knowles, C. Webster, Ann. Clin. Biochem. 47 (2010) 467. [13] S.A. Lagerstedt, D.J. O’Kane, R.J. Singh, Clin. Chem. 50 (2004) 603. [14] W.H.A. de Jong, K.S. Graham, J.C. van der Molen, T.P. Links, M.R. Morris, H.A. Ross, E.G.E. de Vries, I.P. Kema, Clin. Chem. 53 (2007) 1684.

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