Robert L. Taylor and Ravinder J. Singh * Metabolism. Clinical Chemistry 48: (2002) Endocrinology and

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1 Clinical Chemistry 48: (2002) Endocrinology and Metabolism Validation of Liquid Chromatography Tandem Mass Spectrometry Method for Analysis of Urinary Conjugated Metanephrine and Normetanephrine for Screening of Pheochromocytoma Robert L. Taylor and Ravinder J. Singh * Background: Metanephrines are biochemical markers for tumors of the adrenal medulla (e.g., pheochromocytoma) and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastomas). We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the measurement of urinary conjugated metanephrines. Methods: We added 250 ng of d 3 -metanephrine (d 3 -MN) and 500 ng of d 3 -normetanephrine (d 3 -NMN) to 1 ml of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 C water bath, neutralized, and prepared by solidphase extraction. The methanol eluates were analyzed by LC-MS/MS in the selected-reaction-monitoring mode after separation on a reversed-phase amide C16 column. Results: Multiple calibration curves for the analysis of urine MN and NMN exhibited consistent linearity and reproducibility in the range of g/l. Interassay CVs were % at mean concentrations of g/l for MN and NMN. The detection limit was 10 g/l. Recovery of MN and NMN ( g/l) added to urine was %. The regression equation for the LC-MS/MS (x) and colorimetric (y) methods was: y 0.81x (r 0.822; n 110). The equation for the HPLC (x) and LC-MS/MS (y) methods was: y 1.09x 0.05 (r 0.998; n 40). Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, MN *Address correspondence to this author at: Hilton 730, Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN Fax ; singh.ravinder@mayo.edu. Received November 7, 2001; accepted December 17, Conclusions: The sensitivity and specificity of the MS/MS method for urinary conjugated metanephrines offer advantages over colorimetric, immunoassay, HPLC, and gas chromatography mass spectrometry methods because of elimination of drug interferences, high throughput, and short chromatographic run time American Association for Clinical Chemistry Pheochromocytoma is a rare but potentially fatal tumor arising from chromaffin cells, which may produce episodic secondary hypertension along with headaches, sweating, and palpitations (1 4). The incidence of pheochromocytoma has been estimated to be per 1 million persons per year (5, 6). The prevalence of pheochromocytoma was found to be 0.13% for hypertensive patients in a 50-year autopsy study (7) and was estimated to be 6.5% for patients with incidentally discovered adrenal masses. Screening for pheochromocytoma is typically part of an evaluation for secondary causes of hypertension, unexplained spells, incidental adrenal masses, or less commonly, of patients with a family history of pheochromocytoma. Biochemical testing for pheochromocytoma typically has included measurements of 24-h urinary conjugated metanephrines and catecholamines (8). In methods for urinary metanephrines, the conjugated metanephrines are hydrolyzed for analysis because excretion of free metanephrines is negligible. The longer half-lives of sulfateconjugated metanephrines than free catecholamines are consistent with the nature of sulfate-conjugated metanephrines as end products of catecholamine metabolism, the circulatory clearance of which is dependent on elimination by the kidneys (9). High diagnostic sensitivity of plasma metanephrines has been proposed not only because the tumor has the ability to O-methylate catecholamines, but also because 533

2 534 Taylor and Singh: LC-MS/MS Method for Conjugated MN and NMN the half-lives of sulfate-conjugated metanephrine (MN) 1 and normetanephrine (NMN) are longer than those of free catecholamines (10 12). Plasma concentrations of free metanephrines are 5% of the concentrations of the conjugated metanephrines. Recently, measurements of fractionated plasma free metanephrines by HPLC with electrochemical detection (HPLC-EC) were found to have sensitivities and specificities as high as 100% and 89%, respectively, for detecting pheochromocytoma (13, 14). In contrast, the poor sensitivity and specificity of total 24-h urine colorimetric metanephrine method was also reported in the same study. Thus, measurement of fractionated plasma metanephrines has been recommended as the best initial screening test for pheochromocytoma (15). In a 5-year retrospective review and similar studies, analysis of urinary 24-h conjugated MN and NMN has also been found to be an optimum screening test for discriminating secondary causes of hypertension and pheochromocytoma (16). Several methods, including colorimetric, immunoassay, HPLC, and gas chromatography mass spectrometry (GC-MS), have been reported for determination of urinary MN and NMN. Colorimetric assays of urinary total metanephrines have been superseded by HPLC assays that allow separate measurement of MN and NMN, termed fractionated metanephrines. Despite the superiority of HPLC assays for urinary fractionated metanephrines over the colorimetric assay urinary total metanephrines, use of the latter test has persisted (17). Limitations of the colorimetric assay include drug interferences and the lack of an internal standard. Drug interferences are detected in the colorimetric assay when an abnormal spectral curve is generated with the three monitored wavelengths, but some drug and catecholamine interferences can produce normal spectral curves (18). Although new immunoassays for metanephrines have been shown to be free of drug interference (19), they still lack an internal standard to monitor recovery through the extraction process. Recent modifications in HPLC methods have resolved known drug interferences from MN (20), but analytical run times have been lengthened. To overcome drug interferences, an isotope-dilution GC-MS method (21) has also been developed. The GC-MS method is specific, but it requires time-consuming derivatization of the metanephrines before measurement and has longer run times. This study presents a simple high-throughput liquid chromatography tandem MS (LC-MS/MS) method that uses stable deuteriumlabeled isotopes of MN and NMN. 1 Nonstandard abbreviations: MN, metanephrine; NMN, normetanephrine; EC, electrochemical detection; GC-MS, gas chromatography mass spectrometry; LC-MS/MS, liquid chromatography tandem mass spectrometry; E, epinephrine; NE, norepinephrine; D, dopamine; and RO, reverse osmosis. Materials and Methods materials MN and NMN were purchased from Sigma. d 3 -MN was purchased from Cambridge Isotope Laboratories, and d 3 -NMN was purchased from Medical Isotopes. Working solutions of MN and NMN and the stable isotopes were prepared from stock solutions in HCl (0.05 mol/l). Solidphase extraction Oasis cartridges were purchased from Waters Corporation. Analytical discovery LC and guard columns were purchased from Supelco. For the drug interference study, chlorpromazine, desipramine, and ephedrine sulfate were obtained from a local pharmacy. Epinephrine (E), norepinephrine (NE), and dopamine (D) were purchased from Sigma. sample preparation and specimen stability We added 250 ng of d 3 -MN and 500 ng of d 3 -NMN to 1.0 ml of urine, which was then acidified with 50 L of 4.5 mol/l HCl. The urine was then hydrolyzed for 20 min in a boiling water bath. After hydrolysis, the urine was neutralized and the ph adjusted to 6.5 ( 0.5) with 5.0 mol/l NaOH. The urine was then applied to an Oasis HLB extraction cartridge, which was preconditioned with 1.0 ml of methanol and 1.0 ml of reverse-osmosis (RO) H 2 O. After application of the urine, the cartridge was washed with 1.0 ml of RO H 2 O. The MN, NMN, and stable isotopes were eluted from the cartridge with 1 ml of 200 ml/l methanol, transferred to a sealed glass autosampler vial, and injected onto an LC-MS/MS system by an autosampler. For multiple use, the cartridges were washed with 1.0 ml of 200 ml/l methanol, 2.0 ml of 700 ml/l methanol, and 2.0 ml of absolute methanol and reused up to five times. To determine the best preservative for urine collection for analysis of MN and NMN, we collected five random urine samples and separated each sample into three pools. For each sample, one pool was stored with no preservative, one pool was stored with boric acid as the preservative (1 g of boric acid per 20 ml of urine), and one pool was stored with acetic acid as preservative (0.25 ml of a 500 ml/l solution per 20 ml of urine). A 2.0-mL aliquot was immediately taken from each of the pools with or without preservative, labeled as baseline, and frozen. Each pool was divided in two smaller pools, with one stored at room temperature and the other stored in the refrigerator. On days 1, 3, and 7, we removed and froze a 2-mL aliquot from each pool. At the end of the study, all samples were analyzed for fractionated metanephrines. methods Calibrators ( g/l) were prepared in 200 ml/l methanol by dilution of the working solutions of the calibrators and stable isotopes. The 200 ml/l methanol eluate and calibrators were analyzed on an LC-MS/MS system equipped with an API 2000 triple quadrupole mass spectrometer (Sciex). Peripherals included a Perkin- Elmer Series 200 micropump and autosampler. A 15- L

3 Clinical Chemistry 48, No. 3, injection volume was used. Separation was performed on a Discovery RP Amide C16 column ( cm; Supelco). The column was directly connected to the electrospray ionization probe operating at 450 C. The LC-MS/MS method was compared with the colorimetric and HPLC methods for the analysis of urinary conjugated metanephrines for different patients screened for pheochromocytoma. ms/ms conditions The metanephrines were detected in the multiple-reaction monitoring mode of the tandem mass spectrometer with the following transitions: MN, m/z 180 to m/z 148; d 3 -MN, m/z 183 to m/z 151; NMN, m/z 166 to m/z 134; d 3 -NMN, m/z 169 to m/z 137. Data were acquired and processed with the Analyst Software (Ver. 1.1; Sciex). All results were generated in positive-ion mode with the entrance potential at 5 V, the collision cell entrance potential at 10 V, and the cell exit potential at 1.0 V. The optimized declustering potentials were set at 50, 50, 40, and 40 V; the focusing potentials at 380, 380, 350, and 380 V; and the collision energy potential at 30, 30, 30, and 20 V for MN, d 3 -MN, NMN, and d 3 -NMN, respectively, as determined by manual tuning. Front-end electrospray settings for the MS/MS ionization source were as follows: curtain gas, 30; GS1, 90; GS2, 90; CAD, 12, temperature, 450 C; and ion source at 5000 V. For all MS/MS experiments, mass calibration and resolution adjustments [at 0.7 atomic mass units (amu) at full width at half height] on both the resolving quadrupoles were optimized using a polypropylene glycol solution with an infusion pump. Collisionally activated decomposition MS/MS was performed through the closeddesign Q 2 collision cell operating with nitrogen as collision gas. drug interferences Saturated solutions of desipramine, ephedrine sulfate, and chlorpromazine were prepared in 0.05 mol/l HCl. Solutions containing E, NE, and D (100 mg/l of each) were prepared in 0.05 mol/l HCl. A 1.0 g/l acetaminophen stock solution was prepared in methanol. The interference of these drugs was studied in 10 different urine specimens. Each of 10 urine samples was divided into four sets of 1.0-mL aliquots. Nothing was added to the first set of aliquots; 25 L of each of the desipramine, ephedrine sulfate, and chlorpromazine solutions was added to the second set of aliquots; 50 L of the acetaminophen stock solution was added to the third set of aliquots; and 50 L of each of the E, NE, and D solutions was added to the fourth set of aliquots. All samples were extracted and analyzed by the analytically validated LC- MS/MS method. Results lc-ms/ms characteristics of mn and nmn The electrospray MS spectra obtained in the positive-ion mode by infusion of 10 mg/l MN is shown in Fig. 1A. Fig. 1. Electrospray ionization mass spectrum (A) and product ion spectrum (B) for MN. (A), electrospray ionization mass spectrum for MN in positive-ion mode (Q 1 scan). (B), product ion spectrum for the MN m/z 180 ion [M 1 H 2 O]. MN (10 mg/l) in methanol H 2 O 2 mmol/l ammonium acetate was infused at the electrospray tip of the quadrupole mass spectrometer. The first-quadruple (Q 1 ) scan shows a small parent ion peak at m/z 198, which is the expected [M 1] ion, and a larger peak at m/z 180 corresponding to loss of H 2 O from MN [M 1 H 2 O]. Usually, the parent ion at m/z 198 is the preferred parent ion for generating a product ion spectrum. However, to obtain better sensitivity, we chose the intense ion at m/z 180 for the analysis of MN. A similar relationship of a loss of H 2 O was seen for NMN Q 1 scans generating parent ion peaks at m/z 184 and 166 (data not shown). The spectrum in Fig. 1B was acquired by transmitting the MN ion at m/z 180 via Q 1 and scanning for products resulting from fragmentation in the collision cell in the resolving quadruple (Q 3 ). Major daughter ions at m/z 165, 148, and 120 were observed from the fragmentation of the m/z 180 ion peak. Using the autotune algorithm provided in the system software, we optimized the instrument for transmission of

4 536 Taylor and Singh: LC-MS/MS Method for Conjugated MN and NMN NMN and d 3 -NMN coeluted at 1.55 min. Total run time for the analysis was 3 min/sample. Reversed-phase chromatographic analysis of MN and NMN in urine from a pheochromocytoma patient produced a chromatogram similar to the one shown in Fig. 2B. The MN and NMN concentrations were 800 and 1400 g/24 h, respectively, for the pheochromocytoma patient. We separated the major interferences from the urine matrix from MN and NMN by decreasing the methanol in the mobile phase to 150 ml/l, but the decreased methanol concentration substantially reduced the signal-to-noise ratios of the MN and NMN peaks. precision This LC-MS/MS method for the analysis of total urinary MN and NMN was found to be highly precise in the low normal and high abnormal values relevant for the screening and diagnosis of pheochromocytoma. Patient samples at four different concentrations were pooled, aliquoted, and frozen for analysis of MN and NMN. Interassay CVs were 10% for the LC-MS/MS method at concentrations of g/l for MN and NMN. Intraassay CVs were not 13% at concentrations of g/l. The intraand interassay precision data are summarized in Table 1. The detection limit of the assay was 10 g/l for urinary MN and NMN based on an interassay CV 20% for the low-concentration patient pool. Fig. 2. LC-MS/MS chromatograms of a calibrator (A) and urine from a pheochromocytoma patient (B). (A), calibrator contains 200 g/l MN and NMN with stable isotopes d 3 -MN (250 ng) and d 3 -NMN (500 ng). (B), MN (800 g/24 h) and NMN (1400 g/24 h) concentrations were calculated for this patient. the protonated molecular ion, m/z 180, and for maximum intensity of the selected fragment, m/z 148. The product ion at m/z 148 represents a loss of 32 amu from the parent ion, indicating the loss of an OCH 3 group and a proton. The same procedure was used to determine and optimize the Q 1 and Q 3 ions for d 3 -MN, NMN, and d 3 -NMN. Fig. 2A shows LC-MS/MS chromatograms for MN and NMN in a calibrator, each at a concentration of 200 g/l, with retention times of 1.55 and 1.65 min, respectively, and a total run time of 3 min. Signal-to-noise ratios of 31:1 and 42:1 were observed for MN and NMN, respectively, for a 10 g/l calibrator. To maintain the chromatographic sharpness of the peaks for MN and NMN, we used a flow rate of 1 ml/min. The flow was split, with 0.8 ml/min going to the waste and 0.2 ml/min to the tandem mass spectrometer. MN and d 3 -MN coeluted at 1.65 min, whereas recovery Lack of an internal standard is considered one of the major disadvantages of the colorimetric method for the analysis of urinary conjugated MN and NMN (18). In HPLC-EC methods, the recovery of MN and NMN from a patient s urine is normalized with 4-hydroxy-3-methoxybenzylamine hydrochloride, a compound with relatively different chromatographic and electrochemical properties. Both compounds were added at three concentrations in the range of g/l to four patient samples with low endogenous concentrations of MN and NMN and assayed in single determinations. Individual sample recoveries ranged from 92% to 109% and from 91% to 114% for MN and NMN, respectively, in the LC-MS/MS method. Mean recovery data are summarized in Table 2. Table 1. Precision data for the analysis of urinary MN and NMN by LC-MS/MS method. MN NMN Interassay precision Intrassay precision Mean, g/l n CV, % Mean, g/l n CV, %

5 Clinical Chemistry 48, No. 3, Table 2. Mean linearity and recovery characteristics of urinary MN and NMN in the LC-MS/MS method (n 4). Linearity, % observed/expected Recovery, % Dilution MN NMN Added MN and NMN, g/l MN NMN 1: : : : : linearity The LC-MS/MS method for the analysis of MN and NMN was linear at g/l; urine specimens with MN or NMN 5000 g/l can be diluted with water. The linearity data are summarized in Table 2. Four patient samples were assayed in single determinations at several dilutions between 1:2 and 1:40, using RO H 2 O. The expected value of each dilution was calculated based on the result for the undiluted sample. The linearity was evaluated by dividing the observed value of each dilution by the expected value to determine the percentage of the expected result for each dilution. The percentages of the expected results for MN and NMN diluted in RO H 2 O (1:2 to 1:40 dilutions) were % and % for MN and NMN, respectively, for urine specimens containing g/l MN or NMN. Mean recoveries for several dilutions are shown in Table 2. carryover, efficiency of extraction, and specimen stability LC-MS/MS is a high-throughput method, and almost 20 MN and NMN measurements in different urine samples can be made in 1 h. No carryover was observed during multiple injections of patient urine samples when the instrument was run in batch mode. The extraction efficiency of the solid-phase cartridges for multiple use was confirmed by evaluating multiple extractions. Increased concentrations of MN and NMN were added to four patient samples, and each sample was separated into five 1.0-mL aliquots. A cartridge was assigned to each set of aliquots. Each set of aliquots was extracted in its assigned cartridge. Between each extraction, a 1.0-mL aliquot of RO H 2 O with stable isotopes was extracted to monitor carryover. After each extraction, the cartridges were washed with 1.0 ml of 200 ml/l methanol, 2.0 ml of 700 ml/l methanol, and 2.0 ml of absolute methanol. Each sample extract and RO H 2 O extract was analyzed for MN and NMN. The results of the carryover studies are shown in Table 3 and indicate that extraction cartridges can be used up to five times for multiple patients for cost-saving purposes. The conjugated metanephrines were stable under various storage conditions. For urine stored at ambient temperature, day 7 aliquots differed from baseline aliquots, on average, by 4.4%, 0.6%, and 5.8% for MN and Table 3. Extraction efficiency and carryover data for a single cartridge for urinary MN and NMN. Cartridge multiple use Sample MN, g/l Recovery, % NMN, g/l Recovery, % 1st Patient nd Patient rd Patient th Patient th Patient %, 11.5%, and 10.9% for NMN in urine stored without preservative and with boric and acetic as preservative, respectively. For refrigerated storage, day 7 aliquots differed from baseline aliquots by an average of 0.2%, 9.2%, and 3.0% for MN and 0.2%, 3.1%, and 9.0% for NMN in urine stored without preservative and with boric and acetic as preservative, respectively. The results indicate that urine can be collected in any of the above preservatives and shipped frozen to a reference laboratory for analysis without any major loss of conjugated metanephrines. method comparisons Unused portions of 110 specimens analyzed for total metanephrines by a colorimetric method were reanalyzed by the LC-MS/MS method. Only samples with normal spectral curves were used for this study. The correlation between the LC-MS/MS (y) method and the colorimetric method (x) was: y 0.81x (r 0.822). Comparison of the LC-MS/MS vs the colorimetric method showed a mean difference of mg/24 h for total metanephrines with no bias and is shown as a Bland Altman plot in Fig. 3. In a second method comparison, 40 samples were split Fig. 3. Bland Altman plot for comparison of the LC-MS/MS method with the colorimetric Pisano method (n 110). The regression equation for the comparison for total MN is: y 0.81x (r 0.822).

6 538 Taylor and Singh: LC-MS/MS Method for Conjugated MN and NMN Fig. 4. Bland Altman plot for comparison of the LC-MS/MS method with the HPLC-EC method (n 37). Linear regression for all samples (n 40) in the comparison produced the following equation: y 1.09x 0.05 (r 0.998). into two aliquots with one aliquot being analyzed by our LC-MS/MS method (y) and the other aliquot analyzed by the HPLC-EC method (x). The Bland Altman plot (Fig. 4) showed a mean difference of 0.07 mg/24 h for total metanephrines with no bias. The linear regression equation for the correlation was: y 1.09x 0.05 (r 0.998). interferences Recently, the interference of acetaminophen in the HPLC-EC method was addressed by lengthening the chromatographic run time (20). No interference of added acetaminophen (up to 50 mg/l) was observed during the analysis of MN and NMN by the present LC-MS/MS method in a patient urine specimen being screened for the cause of secondary hypertension. Chlorpromazine, desipramine, and ephedrine sulfate are among the drugs known to interfere in the colorimetric or HPLC assays. Chlorpromazine, desipramine, ephedrine sulfate, E, NE, or D added to patient urine samples had no effect on the analysis of MN or NMN by the LC-MS/MS method. Ten different urine samples to which desipramine, ephedrine sulfate, and chlorpromazine had been added had mean differences of 2.3% for MN and 1.8% for NMN compared with the baseline samples. The samples to which acetaminophen had been added had mean differences of 4.2% for MN and 4.1% for NMN compared with baseline. The samples with added E, NE, and D had mean differences of 3.9% for MN and 5.2% for NMN compared with baseline values. Discussion The Q 1 scan in the positive-ion mode for MN shows a small parent ion peak at m/z 198, the expected location of the [M 1] ion, and a larger ion peak at m/z 180. The m/z 180 peak indicates a loss of H 2 O during electrospray ionization in the Q 1 scan [M 1 H 2 O]. [M 1 H 2 O] protonated molecules have also been reported with derivatized catecholamines (22). We chose the m/z 180 parent ion to maximize the sensitivity in our method. A similar relationship for [M 1] and [M 1 H 2 O] was seen in the Q 1 scans of NMN, d 3 -MN, and d 3 -NMN. The acceptable precision, recovery, and linearity data for all of our experiments indicate that this loss of H 2 O in the Q 1 scan from MN and NMN during ionization in electrospray mode is consistent. The shorter run time used to obtain chromatograms and calculate MN and NMN substantially improved turnaround time and sample throughput for the analysis of urine metanephrines compared with the colorimetric and HPLC methods. The use of deuterium-labeled MN and NMN compounds with chromatographic and ionization properties similar to those of MN and NMN is one of the major strengths of the LC-MS/MS method and provides consistent and accurate recoveries. The correlation of the LC-MS/MS method with the colorimetric and HPLC methods was acceptable. The inter- and intraassay CVs were comparable to those for a previously reported urine HPLC method (23). The detection limit for the assay was established as 10 g/l based on a CV of 15% for interassay imprecision at the low end of the concentration range. Several drugs commonly used by patients with hypertension cause interference in colorimetric and HPLC assays. Acetaminophen, for example, has been reported as an interferent in HPLC assays (24) and may require patients to discontinue drug therapy for sample collection. Acetaminophen had no effect on MN or NMN quantification by the LC-MS/MS method. Recently, the HPLC method has eliminated the acetaminophen interference (20), but it requires a longer run time. In general, no major interference was observed during analysis of MN or NMN in urine specimens screened for pheochromocytoma. However, low recovery of the metanephrines was seen in several samples during our validation studies, which was probably attributable to interfering substances in the samples that adsorb to and saturate the binding sites of the cartridge packing. The problem of low recovery in the above circumstances was easily resolved by diluting the samples 1:2 or 1:5 with RO H 2 O. An elution study with the cartridges, where methanol concentrations were varied in 50 ml/l intervals, showed that all MN and NMN was removed from the cartridge at between 50 and 200 ml/l methanol. The 200 ml/l methanol elution removes little of the sample matrix from the cartridge, providing a clean eluate for direct injection into the LC-MS/MS system. It was determined experimentally that two washes (700 ml/l methanol and absolute methanol) were needed to remove the bulk of sample matrix from the cartridge so that the cartridge could be reused. Recovery was consistent throughout the five uses of the cartridge in the carryover study, with no sample-to-sample contamination. Sample ph in two samples was evaluated at ph 4 8 in steps of 1 ph unit to assess optimum sample ph for cartridge extraction. Recovery of the stable isotopes was considerably low at ph 4 or 5, but was acceptable and consistent at ph 6 8. The LC-MS/MS method for urinary conjugated meta-

7 Clinical Chemistry 48, No. 3, nephrines has been validated and implemented in our clinical laboratory and has replaced the colorimetric and HPLC-EC methods. This has improved the analysis turnaround time and also the concern of clinicians about unreliable results reported by the colorimetric assay. The new assay would not require discontinuation of the administered drugs that cause analytical interferences in the colorimetric and HPLC-EC assays. However, drugs known to cause physiologic increases in catecholamines or metanephrines would need consideration during the evaluation of pheochromocytoma. We are participating in an Institution Review Boardapproved study to determine the specificity of the urinary MN and NMN LC-MS/MS method with 100% sensitivity for the detection of pheochromocytoma. The LC-MS/MS method also has the potential to measure fractionated plasma free metanephrines, which will provide analytical advantages similar to those discussed for urinary metanephrines. At present we are developing an efficient extraction and chromatographic procedure with detection limits in the range of ng/l for plasma free metanephrines. It is very likely that in the future, with the implementation of LC-MS/MS methodology in an endocrine laboratory, the analysis of plasma free metanephrines and urinary conjugated metanephrines may be the best tests for biochemical evidence of pheochromocytoma. Several studies have already suggested poor specificity of urine catecholamines and vanillylmandelic acid tests for the workup of pheochromocytoma patients. The LC- MS/MS methods for metanephrines will not only prevent expensive follow-up imaging studies by eliminating false positives reported with the previous methods, but will also allow analysis without taking patients off drugs necessary for management of hypertension. In conclusion, this assay is accurate, precise, and linear for the measurement of urinary MN and NMN and has relatively high throughput. It offers advantages over colorimetric, immunoassay, HPLC, and GC-MS methods in regard to interferences, deuterated internal standard, sample turnaround time, and lack of derivatization. These analytical advantages indicate that this method may be used as a reference method and may provide a tool for the development of proficiency testing material. References 1. Benowitz NL. Pheochromocytoma. Adv Intern Med 1990;35: Weinkove C. Hypertension and the adrenal medulla. Commun Lab Med 1992;3: Bravo EL. Evolving concepts in the pathophysiology, diagnosis, and treatment of pheochromocytoma. Endocr Rev 1994;15: Stenstron G, Haljamae H, Tisell LE. Influence of preoperative treatment with phenoxybenzamine on the incidence of adverse cardiovascular reactions during the anesthesia and surgery for pheochromocytoma. Acta Anaesthesiol Scand 1985;29: Anderson GS, Tofdahl DB, Lund JO, Strandgaard S, Nielsen PE. 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