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Clinical Chemistry 48: 332-337, 2002;
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(Clinical Chemistry. 2002;48:332-337.)
© 2002 American Association for Clinical Chemistry, Inc.

Rapid Analysis of Metanephrine and Normetanephrine in Urine by Gas Chromatography-Mass Spectrometry

David K. Crockett1a, Elizabeth L. Frank2 and William L. Roberts2

1 ARUP Laboratories, Inc., 500 Chipeta Way, Salt Lake City, UT 84108.

2 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132.

aAuthor for correspondence. E-mail crockedk{at}aruplab.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Widely used HPLC methods for quantification of metanephrine and normetanephrine in urine often have long analysis times and are frequently plagued by drug interferences. We describe a gas chromatography-mass spectrometry method designed to overcome these limitations.

Methods: Metanephrine and normetanephrine conjugates were converted to unconjugated metanephrine and normetanephrine by acid hydrolysis. To avoid the rapid decomposition of the deuterated internal standards (metanephrine-d3 and normetanephrine-d3) under hydrolysis conditions, the internal standards were added after hydrolysis. Solid-phase extraction was used to isolate the hydrolyzed metanephrines from urine. Samples were concentrated by evaporation, then derivatized simultaneously with N-methyl-N-(trimethylsilyl)trifluoroacetamide and N-methyl-bis-heptafluoro-butryamide at room temperature.

Results: The assay was linear from 25 to 7000 µg/L. The intraassay CVs were <5% and the interassay CVs <12%. Comparison with a routine HPLC method (n = 192) by Deming regression yielded a slope of 1.00 ± 0.02 µg/L, an intercept of -5.8 ± 7.8 µg/L, and Sy|x = 50.6 µg/L for metanephrine and a slope of 0.94 ± 0.03, intercept of 19 ± 11 µg/L, and Sy|x = 60 µg/L for normetanephrine. The correlation coefficients (r) were calculated after log transformation of the data and gave r = 0.97 for metanephrine and r = 0.97 for normetanephrine. Interference from common medications or drug metabolites was seen in <1% of samples. The time between sequential injections was <7 min.

Conclusions: This new gas chromatography-mass spectrometry assay for total fractionated metanephrines is rapid, compares well with a standard HPLC assay, and avoids most drug interferences that commonly affect HPLC assays for urine metanephrines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Quantification of urine metanephrine and normetanephrine is commonly requested in the diagnosis and monitoring of patients with pheochromocytoma and related neurogenic tumors. The determination of urinary metanephrines is considered a reliable screening test for pheochromocytoma (1)(2). More than 97% of urinary metanephrines are conjugated as either the glucuronide or sulfate forms (3). For measuring total fractionated metanephrines, acid hydrolysis or enzymatic treatment is necessary before the assay. Recent studies indicate that the measurement of plasma metanephrines, either total or free, is an alternative to urinary measurements (4)(5)(6). Plasma metanephrines may be a more sensitive indicator of the presence of a tumor, whereas urinary metanephrines may be more specific (4).

A literature search revealed that most published laboratory methods for metanephrine and normetanephrine analysis in urine use HPLC. These HPLC methods can accommodate simplified sample preparation, but often have relatively long analysis times and are plagued by drug interferences (7)(8)(9). In addition, none of the many methods available are ideally suited for the high volume of samples encountered in a commercial reference laboratory (10)(11)(12)(13). Our goal was to overcome these limitations of current HPLC methods. Consequently, we have developed a rapid and specific method for measurement of total urinary metanephrine and normetanephrine that uses gas chromatography-mass spectrometry (GC-MS). 1


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals and reagents
Metanephrine and normetanephrine were obtained from Sigma. The deuterated internal standards metanephrine-d3 and normetanephrine-d3 were purchased from Medical Isotopes. All other solvents were reagent grade and obtained from Fisher Scientific. The Lyphocheck® urine calibrator and controls were purchased from Bio-Rad Clinical Division.

samples
Urine samples (n = 192), for which metanephrine testing had been performed by HPLC, were chosen at random. Concentrations of metanephrine and normetanephrine ranged from 25 µg/L to 7000 µg/L. All samples were stored at 2–8 °C until analysis. The calibrator and controls were prepared as needed and stored at 2–8 °C. All studies using samples from patients were approved by the Institutional Review Board of the University of Utah Health Sciences Center.

apparatus
Solid-phase extraction was performed with a Speedisk® 48 Pressure Processor (SPEware, Inc.). A TurboVapTM LV Evaporator sample concentrator station from the Zymark Corporation was used to evaporate solvents. Clean Screen® CSDAU 303 solid-phase extraction columns (C8 mixed-mode cation exchange) were purchased from United Chemical Technologies, Inc. The analysis of metanephrines was performed using a Finnigan Voyager GC-MS instrument equipped with an A220S autosampler and ToxLabTM software (Thermoquest). We performed data acquisition in the selected-ion monitoring mode using electron ionization with the detector at 350 mV. The GC was equipped with a DB-5ms capillary column (length, 15 m; diameter, 0.25 mm; film thickness, 0.25 µm; J&W Scientific). The instrument was tuned with perfluro-tributylamine.

assay procedures
A 1.0-mL aliquot sample was used for hydrolysis and extraction. HCl (50 µL of 6.0 mol/L) was added to each urine sample. The tubes were incubated at 90 °C in a dry heat block for 25 min and allowed to cool before proceeding. Internal standard (100 µL containing 5 ng/µL each of metanephrine-d3 and normetanephrine-d3) was added. Solid-phase extraction was performed in the mixed-mode Clean Screen column. The extraction column was preconditioned with 3 mL of methanol followed by 3 mL of water. The samples were loaded and the columns washed with 3 mL of water followed by 3 mL of 0.1 mol/L HCl and then 3 mL of methanol. The columns were dried for 5 min before the metanephrines were eluted. The elution solvent for each column was 3 mL of methanol containing 20 mL/L ammonium hydroxide. The samples were evaporated to dryness and derivatized with 50 µL of acetonitrile, 25 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and 25 µL of N-methyl-bis-heptafluorobutryamide (MBHFBA). The dual derivatization was performed simultaneously at room temperature.

A sample volume of 1 µL was injected with a split ratio of 40:1. The helium carrier gas was set at a constant flow of 2.0 mL/min. The injection port temperature was 200 °C. The initial oven temperature was 120 °C, held for 0.2 min, then ramped at 30 °C/min to 200 °C and held for 0.2 min, then ramped at 70 °C/min to 310 °C and held for 2.0 min. The detector interface temperature was 300 °C. The source temperature was 240 °C. The ions monitored were m/z 508, 509, 511, 512, 522, 523, 525, and 526.

The urine calibrator was prepared according to the package insert and used for single-point calibration. The calibrator target concentrations were 328 and 718 µg/L for metanephrine and normetanephrine, respectively. The negative control was prepared by the addition of 100 µL of working internal standard to blank urine. The blank urine was prepared by diluting a nonpathologic urine sample with water at a ratio of 1:5. The Lyphocheck normal control sample was prepared per package insert directions and used as the low control. The target ranges were 54–90 µg/L for metanephrine and 204–292 µg/L for normetanephrine. The Lyphocheck normal control sample was used as the high control, but prepared at twice the concentration prescribed by the package insert. The target ranges were 400–900 µg/L for metanephrine and 1400–2000 µg/L for normetanephrine. This allowed the controls to bracket the concentration of the single-point calibrator.

The HPLC method uses a 3.0-mL aliquot sample. HCl (2.0 mL of 0.6 mol/L solution) was added to each sample, and the tubes were incubated in a hot oil bath at 107 °C for 30 min and allowed to cool before proceeding. Water (5 mL) was added to each tube, and the samples were filtered and centrifuged for 10 min at 2000g. An aliquot of 1 mL was transferred to each autosampler vial, and 40 µL was injected for analysis. The same calibrator and Lyphocheck controls described above were used, but no internal standard was used. The HPLC method uses the Coulochem® Electronic Array System (ESA Inc.). Mobile phase (no. 70-3067) and HPLC column (no. 70-0340) from ESA Inc. were also used. The limit of detection for this assay was 20 µg/L. The overall imprecision of the assay was 12% for the Lyphocheck normal control sample and 8.3% for the Lyphocheck abnormal control sample. The assay was derived from the method described by Gamache et al. (14).

Nine concentrations (25, 50, 100, 250, 500, 1000, 1500, 3000, and 7000 µg/L) of calibrators were analyzed in triplicate with one run per day for 3 days to assess linearity and imprecision. The long-term stability of the derivatized sample was monitored for 14 days. Sample vials were stored at room temperature. Hydrolysis of the conjugated metanephrine and normetanephrine in urine was optimized by evaluating differing conditions of time, temperature, and acid concentration. EP Evaluator-CLIA software (David G. Rhoads Associates) was used for all regression analysis and calculation of Sy|x and correlation coefficients (r).

All drugs known to interfere with the current HPLC method, including several biogenic amines and cardiac medications, were derivatized and injected to determine retention times and prominent mass fragments. For each compound, 10 mg was derivatized at room temperature with 50 µL of acetonitrile plus 25 µL of MSTFA plus 25 µL of MBHFBA. Normetanephrine-d3 was used to calculate the relative retention times of all other compounds.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The concentration of HCl is key to successful hydrolysis of the metanephrine and normetanephrine conjugates in urine (Fig. 1 ). Increasing concentrations of HCl decomposed the deuterated internal standard. Excessive acid will destroy metanephrine and normetanephrine as well. HCl was tested from 0.1 to 6 mol/L. Time and temperature play a secondary, but minor role in the hydrolysis process. Temperatures between 25 and 120 °C were evaluated. Hydrolysis times from 10 to 120 min were evaluated as well (data not shown). Hydrolysis in 0.3 mol/L HCl at 90 °C for 25 min was optimal. Because of the rapid decomposition of the deuterated internal standards, these compounds were added after the hydrolysis step and before solid-phase extraction.



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  		Figure 1. Evaluation of acid hydrolysis with increasing concentrations of HCl for 25 min at 90 °C.

(A), recovery of metanephrine ({square}) and metanephrine-d3 ({triangleup}) after acid hydrolysis. (B), recovery of normetanephrine ({square}) and normetanephrine-d3 ({triangleup}) under the same hydrolysis conditions.

Linearity was evaluated with nine concentrations analyzed in triplicate. One run a day for 3 days was performed. The linearity of metanephrine and normetanephrine after hydrolysis and solid-phase extraction is shown in Fig. 2 . The limit of detection of the assay, defined as a signal-to-noise ratio >5:1, was 10 µg/L. The limit of quantification, with requirements of accuracy within 85–115% of the target concentration and ion mass ratios within 20% of the target ratio as set by the calibrator, was 25 µg/L. The linear range of the assay was 25–7000 µg/L. This range is consistent with the more widely used HPLC methods. The total-ion chromatogram and full-scan mass spectra of derivatized metanephrine and normetanephrine are shown (Fig. 3 ).



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  		Figure 2. Summary of nine different concentrations (25, 50, 100, 250, 500, 1000, 1500, 3000, and 7000 µg/L) analyzed in one run per day for 3 days.

Linearity after acid hydrolysis and solid-phase extraction for metanephrine (A; y = 0.947x - 7.21; r = 0.999) and normetanephrine (B; y = 0.932x + 35.1; r = 0.999).



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  		Figure 3. GC-MS analysis of urine metanephrines showing the total ion chromatogram from a representative injection in selected-ion monitoring mode (A) and the full-scan mass spectra of metanephrine (B) and normetanephrine (C) derivatized with MSTFA and MBHFBA.

All CVs for within-run imprecision were <4.2%. All CVs for between-run imprecision were <12%. Total imprecision was <12% (Table 1 ). By the addition of deuterated analogs, the GC-MS assay imprecision was greatly improved in contrast to the HPLC method without internal standard.


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  		Table 1. Precision1 of Bio-Rad urine calibrator and controls.

A correlation study was performed using 192 patient samples that were assayed previously for metanephrine and normetanephrine by HPLC. Sample concentrations ranged from 25 to 7000 µg/L. Results from the new GC-MS method were compared with the routine HPLC method (Fig. 4 ). Deming regression was performed before log transformation. Because the data were not gaussian, the Pearson correlation coefficients (r) were calculated after log transformation. Because of interference in the original HPLC method, the results for three metanephrine samples and five normetanephrine samples were not usable, and the data were excluded from the graphs in Fig. 4 . The dual derivative showed excellent stability, with no decrease in the abundance of metanephrine or normetanephrine after 14 days at room temperature.



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  		Figure 4. Comparison of HPLC and GC-MS methods in 186 samples.

Deming regression analysis before log transformation gives a slope of 1.00 ± 0.02, an intercept of -6 ± 8 µg/L, Sy|x = 51 µg/L, and r = 0.97 for metanephrine (A) and a slope of 0.94 ± 0.03, an intercept of 19 ± 11 µg/L, Sy|x = 60 µg/L, and r = 0.97 for normetanephrine (B). Pearson correlation coefficients (r) were calculated after log transformation.

An extensive interference study was performed to evaluate the specificity of the assay. The results are summarized in Table 2 . In addition, of the 192 samples analyzed by GC-MS, only 1 showed interference. The sample contained a coeluting peak next to metanephrine with <40% resolution and ion mass ratio failure. Quantification for this sample was likely affected. Overall, the sample failure rate attributable to poor chromatography or drug interference with either metanephrine or normetanephrine was reduced from 11% (21 of 192 by HPLC) to <1% (1 of 192 by GC-MS).


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  		Table 2. Summary of drug interferences, relative retention times, and mass fragments.

An evaluation of potential column carryover found 1.3 µg/L metanephrine and 7.7 µg/L normetanephrine after a supplemented sample (10 000 µg/L) was injected. As an additional safeguard to prevent carryover, the software is programmed to automatically inject a solvent blank when a concentration threshold of 5000 µg/L is exceeded.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Acid hydrolysis is required before solid-phase extraction to determine the total concentration of metanephrine and normetanephrine. The ideal internal standards would be the deuterated sulfate conjugates of metanephrine and normetanephrine. However, these compounds are not commercially available and are difficult to synthesize in sufficient quantities for routine use. Therefore, they were not used in our study. When the optimal conditions for the hydrolysis of the metanephrine and normetanephrine conjugates are used, the "unprotected" or nonconjugated internal standard is quickly degraded. Therefore, to avoid rapid decomposition of the deuterated analogs under hydrolysis conditions, we added the internal standards after acid hydrolysis. This improved the agreement between GC-MS results and the current well-established reference intervals (1)(9).

Some of the advantages of solid-phase extraction technology are well known (15). However, a cleaner final extract is often exchanged for increased sample preparation time and the additional cost of the solid-phase columns and an extraction manifold. After evaluation of several solid-phase methods and extraction columns, the UCT Clean Screen solid-phase extraction column was selected for its cleanliness and relative low cost. In addition, the extraction was moved to a 48-place positive pressure manifold to increase the efficiency of the extraction.

Routine HPLC methods may take 20 min or longer per injection (5)(7)(8)(9). Our initial experiments showed promise with a much faster GC oven method and a total analysis time of <4 min. However, the ion mass ratios for an occasional specimen would fall outside of the allowed range or an interfering peak would coelute with metanephrine or normetanephrine. Therefore, the oven program was optimized to allow sufficient GC separation of all interfering compounds. This GC-MS method allows a throughput of approximately eight samples per hour in comparison with three per hour by HPLC with a greatly decreased occurrence of interferences. In addition, the cost of a GC-MS system may be as little as 25% more than the HPLC equipped with electrochemical detection. Furthermore, although a higher degree of operator training may be necessary, one GC-MS instrument can replace two HPLC instruments.

The limit of quantification of this assay compares favorably with HPLC methods for urinary metanephrine and normetanephrine quantification. The initial sample aliquot volume, concentration of internal standard, final solvent volume in the autosampler vial, GC injection volume, and injection port split ratio settings can all impact the overall sensitivity of the assay. By adjusting these factors in the assay, we found that the sensitivity could be enhanced. It is important to note, however, that clinically significant concentrations of metanephrines are usually 5- to 10-fold higher concentrations than the upper limit of the reference interval (1). The extended linear range of the assay is a tradeoff for slightly reduced low-end sensitivity. The assay and instrumentation, as described, are not sensitive enough to measure free urinary metanephrines, which are present at only 3% of the total concentration (3).

The double derivatization has several advantages compared with MSTFA alone. The higher molecular weight of the parent fragments (m/z 522 for metanephrine and m/z 508 for normetanephrine) improved sensitivity and decreased interferences. Single peaks were seen for metanephrine and normetanephrine, in contrast to multiple peaks when biogenic amines are derivatized with MSTFA only. This enhanced sensitivity because only one peak was present for each compound. In addition, when drugs that could interfere with HPLC methods were tested, no interference was found. No acid anhydride byproducts are produced when MBHFBA is used, as are common in derivatization with pentafluoropropionic acid anhydride or heptafluorobutyric acid anhydride. This eliminated a second evaporation and reconstitution step and extended the useful lifetime of the GC column because acid derivatives were not injected. The two derivatization reactions can be performed simultaneously, without heated incubation. This dual derivatization technique not only improves chromatography and decreases interference, but also enhances the sensitivity.

In moving the assay from HPLC to GC-MS, several improvements were made. The sample aliquot was decreased from 3 mL to 1 mL, allowing the laboratory sufficient volume to retest the specimen if necessary. Deuterated internal standards were incorporated, leading to improved accuracy and precision. Interferences from drugs and drug metabolites decreased from 11% to <1%. Carryover from samples with increased metanephrine or normetanephrine concentrations was minimal. The assay linearity was extended, thereby decreasing the number of repeat analyses for concentrations above the linear range. Analysis time was decreased from 22 min per sample to 7 min per sample. This assay is fast and simple, is reliable in a high-volume clinical laboratory setting, and to date, analysis of more than 8000 samples has been performed.


   Acknowledgments
 
This work was supported by the ARUP Institute for Clinical and Experimental Pathology.


   Footnotes
 
1 Nonstandard abbreviations: GC-MS, gas chromatography-mass spectrometry; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; and MBHFBA, N-methyl-bis-heptafluorobutryamide.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rosano TG, Whitley RJ. Catecholamines and serotonin. Burtis CA Ashwood ER eds. Tietz textbook of clinical chemistry 3rd ed. 1999:1570-600 WB Saunders Philadelphia. .
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