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Background: Measurements of plasma or urinary metanephrines are recommended for diagnosis of pheochromocytoma and paraganglioma (PPGL). What test offers optimal diagnostic accuracy for patients at high and low risk of disease, whether urinary free metanephrines offer advantages over deconjugated metanephrines, and what advantages are offered by including methoxytyramine in panels all remain unclear., Methods: A population of 2056 patients with suspected PPGLs underwent prospective screening for disease using mass spectrometric-based measurements of plasma free, urinary deconjugated, and urinary free metanephrines and methoxytyramine. PPGLs were confirmed in 236 patients and were excluded in others on follow-up evaluation., Results: Measurements of plasma free metabolites offered higher ( P < 0.01) diagnostic sensitivity (97.9%) than urinary free (93.4%) and deconjugated (92.9%) metabolites at identical specificities for plasma and urinary free metabolites (94.2%) but at a lower ( P < 0.005) specificity for deconjugated metabolites (92.1%). The addition of methoxytyramine offered little value for urinary panels but provided higher ( P < 0.005) diagnostic performance for plasma measurements than either urinary panel according to areas under ROC curves (0.991 vs 0.972 and 0.964). Diagnostic performance of urinary and plasma tests was similar for patients at low risk of disease, whereas plasma measurements were superior to both urinary panels for high-risk patients., Conclusions: Diagnosis of PPGLs using plasma or urinary free metabolites provides advantages of fewer false-positive results compared with commonly measured deconjugated metabolites. The plasma panel offers better diagnostic performance than either urinary panel for patients at high risk of disease and, with appropriate preanalytics, provides the test of choice. Measurements of methoxytyramine in urine show limited diagnostic utility compared with plasma., (© 2018 American Association for Clinical Chemistry.)

Background: Pheochromocytomas are rare catecholamine-producing tumors derived in more than 30% of cases from mutations in 9 tumor-susceptibility genes identified to date, including von Hippel-Lindau tumor suppressor (VHL); succinate dehydrogenase complex, subunit B, iron sulfur (Ip) (SDHB); and succinate dehydrogenase complex, subunit D, integral membrane protein (SDHD). Testing of multiple genes is often undertaken at considerable expense before a mutation is detected. This study assessed whether measurements of plasma metanephrine, normetanephrine, and methoxytyramine, the O-methylated metabolites of catecholamines, might help to distinguish different hereditary forms of the tumor., Methods: Plasma concentrations of O-methylated metabolites were measured by liquid chromatography with electrochemical detection in 173 patients with pheochromocytoma, including 38 with multiple endocrine neoplasia type 2 (MEN 2), 10 with neurofibromatosis type 1 (NF1), 66 with von Hippel-Lindau (VHL) syndrome, and 59 with mutations of SDHB or SDHD., Results: In contrast to patients with VHL, SDHB, and SDHD mutations, all patients with MEN 2 and NF1 presented with tumors characterized by increased plasma concentrations of metanephrine (indicating epinephrine production). VHL patients usually showed solitary increases in normetanephrine (indicating norepinephrine production), whereas additional or solitary increases in methoxytyramine (indicating dopamine production) characterized 70% of patients with SDHB and SDHD mutations. Patients with NF1 and MEN 2 could be discriminated from those with VHL, SDHB, and SDHD gene mutations in 99% of cases by the combination of normetanephrine and metanephrine. Measurements of plasma methoxytyramine discriminated patients with SDHB and SDHD mutations from those with VHL mutations in an additional 78% of cases., Conclusions: The distinct patterns of plasma catecholamine O-methylated metabolites in patients with hereditary pheochromocytoma provide an easily used tool to guide cost-effective genotyping of underlying disease-causing mutations.

Background: Measurements of 24-h fractionated urinary metanephrines and catecholamines are used for the diagnosis of pheochromocytoma, but adequate information is needed regarding collection, storage, and shipment conditions., Methods: Spot urine samples were collected from 8 healthy volunteers. Aliquots were immediately frozen at -20 degrees C, or acidified to pH 4 and then frozen either directly or after 24 h at room temperature. The remaining urine was left at room temperature for 24 h and then split into one portion that was acidified and one portion that was not. Aliquots were either frozen or allowed to stand at room temperature for an additional 24, 48, 72, 96, and 168 h before freezing. We also tested the efficacy of adding Na(2)EDTA and Na(2)S(2)O(5), as an alternative to acidification for preservation of the catecholamines., Results: No clinically relevant degradation (<5%) was observed for the fractionated metanephrines under any of the storage conditions. In contrast, in approximately 50% of the untreated samples catecholamines were partially degraded during the first 24 h at room temperature. Immediate acidification, however, prevented degradation, whereas acidification after 24 h prevented further decay. Addition of Na(2)EDTA and Na(2)S(2)O(5) fully prevented degradation of catecholamines during the first 24 h in 4 of 5 cases. In the remaining case, degradation did not exceed 10%., Conclusion: Preservation of samples for measurements of urinary fractionated metanephrines is not necessary if samples are assayed or frozen within 1 week, which is an important advantage if transport of samples is necessary. In contrast, urinary catecholamines require preservation measures during collection.

Background: Despite the rarity of pheochromocytoma, diagnosis is important because of the dangers of uncontrolled severe hypertension and the availability of very effective surgical treatment. Urinary or plasma catecholamines or catecholamine derivatives are commonly used to screen for pheochromocytomas before imaging, but data from 24-h urinary metanephrine results, patient age, and sex may better predict tumors in populations with a low pretest probability., Methods: We retrospectively studied outcomes of an unselected population (1819 patients) referred to a tertiary hospital laboratory for urinary metanephrine testing and investigated the usefulness of some simple derivative measures for detecting pheochromocytoma. We normalized values for urinary 24-h excretion of metanephrine, normetanephrine, and 3-methoxytyramine by dividing by an age- and sex-specific reference range. We then compared pheochromocytoma prediction by the use of products of these normalized measures with the gold standard of biopsy-confirmed tumor., Results: The product of the excretion of normalized metanephrine (nMAD) and normalized normetanephrine (nNMT) (nMAD.nNMT) was a highly sensitive (100%) and specific (99.1%) measure, yielding a positive predictive value of 82%. ROC curves were not improved by including the normalized 3-methoxytyramine concentrations in the product. The test for nMAD.nNMT gave higher sensitivity and specificity than the tests for either substance alone., Conclusion: The test for nMAD.nNMT is a useful measure for identifying pheochromocytoma in a population with a low pretest probability.

Background: Measurements of plasma free metanephrines (normetanephrine and metanephrine) provide a useful test for diagnosis of pheochromocytoma and may provide other information about the nature of these tumors., Methods: We examined relationships of tumor size, location, and catecholamine content with plasma and urinary metanephrines or catecholamines in 275 patients with pheochromocytoma. We then prospectively examined whether measurements of plasma free metanephrines could predict tumor size and location in an additional 16 patients., Results: Relative proportions of epinephrine and norepinephrine in tumor tissue were closely matched by relative increases of plasma or urinary metanephrine and normetanephrine, but not by epinephrine and norepinephrine. Tumor diameter showed strong positive relationships with summed plasma concentrations or urinary outputs of metanephrine and normetanephrine (r = 0.81 and 0.77; P <0.001), whereas relationships with plasma or urinary catecholamines were weaker (r = 0.41 and 0.44). All tumors in which increases in plasma metanephrine were >15% of the combined increases of normetanephrine and metanephrine either had adrenal locations or appeared to be recurrences of previously resected adrenal tumors. Measurements of plasma free metanephrines predicted tumor diameter to within a mean of 30% of actual diameter, and high plasma concentrations of free metanephrine relative to normetanephrine accurately predicted adrenal locations., Conclusions: Measurements of plasma free metanephrines not only provide information about the likely presence or absence of a pheochromocytoma, but when a tumor is present, can also help predict tumor size and location. This additional information may be useful for clinical decision-making during tumor localization procedures.

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 d3-metanephrine (d3-MN) and 500 ng of d3-normetanephrine (d3-NMN) to 1 mL of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 degree C water bath, neutralized, and prepared by solid-phase 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 10-5000 microg/L. Interassay CVs were 5.7-8.6% at mean concentrations of 90-4854 microg/L for MN and NMN. The detection limit was 10 microg/L. Recovery of MN and NMN (144-2300 microg/L) added to urine was 91-114%. The regression equation for the LC-MS/MS (x) and colorimetric (y) methods was: y = 0.81x - 0.006 (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)., 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.

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-d(3) and normetanephrine-d(3)) 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 microg/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 microg/L, an intercept of -5.8 +/- 7.8 micro/L, and S(y/x) = 50.6 microg/L for metanephrine and a slope of 0.94 +/- 0.03, intercept of 19 +/- 11 microg/L, and S(y/x) = 60 microg/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.

Background: Determination of urinary free catecholamine and total (i. e., free plus conjugated) metanephrine excretion is considered the most clinically sensitive biochemical test for pheochromocytoma. In this study, we evaluated new immunoassay methods for the measurement of these analytes for potential drug-based interference., Methods: Urine samples collected from patients on a variety of medications were grouped by specific drug type. The significance of any difference in the free catecholamine or total metanephrine concentrations in the different groups was assessed by one-way ANOVA. A group of patients receiving no medication was included as a control (no analytical interference). Additionally, analytical accuracy, detection limit, and precision were determined., Results: No significant differences were found in the concentrations of free catecholamines or total metanephrines in urine from patients taking the medications investigated and the control group: P = 0.649 (fE), 0.221 (fNE), 0.149 (tM), and 0.170 (tNM). For free catecholamines, intraassay CVs were 4.6-18%; interassay CVs were 10-25%. For total metanephrines, intraassay CVs were 9.6-27%; interassay CVs were 5. 8-22%. Detection limits were 0.009 and 0.027 micromol/L for fE and fNE and 0.119 and 0.346 micromol/L for tM and tNM, respectively., Conclusions: None of the drugs examined in this study interfered in the measurement of free catecholamines or total metanephrines by these immunoassays. The technique is easier to use, requires less equipment, and is more accessible than HPLC. In combination, these assays are suitable as initial screening tests for pheochromocytoma.

Determination of urinary 3-O-methylated catecholamines (metanephrines) is generally considered a principal test for the clinical chemical diagnosis of pheochromocytoma and is currently performed predominantly with chromatographic techniques such as gas-liquid chromatography and HPLC. Enzyme immunoassays based on microtiter plate technology have recently been developed for the quantitative determination of urinary metanephrine (M) and normetanephrine (NM). We compared the results for urinary M and NM determined by these ELISA methods with those obtained by a recently developed isotope dilution mass spectrometric method. From this comparative study we can conclude that the investigated ELISA methods are applicable in the quantification of urinary M and thus can be successfully used to establish the diagnosis of pheochromocytoma. These relatively simple methods can be executed in any clinical laboratory and in time may replace the present, more complicated, chromatographic techniques.

We review our data on the measurement of catecholamines and their metabolites in 19 patients with pheochromocytoma. All the assays were specific high-performance liquid chromatographic procedures with electrochemical detection. The assay of fractionated metanephrines was 100% sensitive. Normal values for both urinary norepinephrine and epinephrine were found in two asymptomatic patients with pheochromocytoma. Normal values for 3-methoxy-4-hydroxymandelic acid (VMA) were found in two patients with pure epinephrine-secreting tumors and in one patient with multiple endocrine neoplasia type II. Plasma catecholamines were usually less increased than their urinary counterparts. We recommend the specific measurement of norepinephrine and epinephrine as the initial test for patients with suggestive symptoms, and specific measurement of normetanephrine and metanephrine for patients in whom an adrenal mass is incidentally found. We argue against the use of total metanephrines, total catecholamines, and VMA because of their lack of diagnostic sensitivity.

We describe a procedure for the direct measurement of metanephrine (MN) and normetanephrine (NMN) in hydrolyzed urine, using HPLC with coulometric array detection. Acid-hydrolyzed samples were diluted and filtered before separation by isocratic reversed-phase ion-pair chromatography. Eight serial coulometric sensors, set at incrementally increasing anodic potentials, were used to screen lower-oxidizing interferences and provide stepwise oxidation of the metanephrines. Voltammetric behavior across three adjacent sensors was used to assess resolution and aid in peak identification. Values obtained in commercial controls were consistently within the specified target range. Variability, expressed as CV, was 5.45-9.22% between runs and 1.60-4.52% within-run for both compounds. The limit of detection was 2.6 micrograms/L for MN and 2.8 micrograms/L for NMN, with a linear response to 15.0 mg/L for both analytes. Results from patients' samples correlated well with those by a method involving dual ion-exchange extraction (r = 0.963, n = 82 for MN; r = 0.9768, n = 83 for NMN). This procedure provided high selectivity and objective peak purity information while greatly simplifying sample preparation.

The urinary excretion of normetanephrine, metanephrine, and 3-methoxy-4-hydroxymandelic acid was quantified by HPLC in hypertensive patients who were being routinely investigated for the exclusion of pheochromocytoma. The data were used to calculate reference intervals for these analytes. Age- and sex-specific 95% reference intervals for 24-h urinary excretion were as follows (mumol/24 h): normetanephrine in men ages 16-35 years (n = 17) 0.7-3.4, in men older than 35 years (n = 121) 0.8-5.1, in women ages 16-35 years (n = 41) 0.5-2.1, and in women older than 35 years (n = 144) 0.6-3.3 mumol/24 h; metanephrine in men (n = 138) 0.3-2.0, and in women (n = 185) 0.2-1.3; and 3-methoxy-4-hydroxymandelic acid in men (n = 142) 10-54, and in women (n = 184) 9-38. Normetanephrine or metanephrine excretion rates or both exceeded the proposed reference intervals in seven of the eight patients with pheochromocytoma. Determination of 3-methoxy-4-hydroxymandelic acid excretion did not yield any additional diagnostic information.

In this relatively simple procedure for extracting metanephrines from urine, after an internal standard (4-hydroxy-3-methoxybenzylamine in 1 mmol/L HCl) is added, the sample is hydrolyzed in a boiling water bath, then treated with ammonia and alumina. Excess ammonia is removed under reduced pressure and the sample is applied to a 1-mL Bond Elut SCX column, which is washed, and metanephrines and internal standard are eluted with 0.5 mmol/L sodium acetate/acetonitrile (3/1 by vol). Of this elute, 5 microL is injected into a 15 cm x 4.6 mm (i.d.) column packed with 5-microns octadecylsilyl silica particles, which is eluted with a mobile phase containing tetramethylammonium perchlorate. Peaks are detected coulometrically at +0.28 V. In the resulting chromatogram, metanephrines give sharp peaks, well resolved from peaks for solvent and internal standard. There are no extraneous peaks for catechols or mono-oxygenated phenylethylamines. Results correlated well (r = 0.999, n = 13) with those by earlier described liquid-chromatography.

Hydrolyzed urine with added ring-trideuterated normetanephrine and metanephrine is applied to wet C18-reversed-phase minicolumns. The "metanephrines" are eluted, dried, derivatized with pentafluoropropionic anhydride, and analyzed with the gas chromatograph-mass spectrometer. Ions for the nondeuterated and trideuterated compounds are monitored at ml z 458 and 461, respectively. For both normetanephrine and metanephrine, the standard curve is linear over the range 10-2000 micrograms/L and the procedure has adequate precision both within-run (CV less than 3%) and between-day (CV less than 7%). Alkaline pH in the extraction is important for optimal analytical recovery. We have examined the potential value of untimed urine specimens for screening purposes and compared 24-h urine concentrations of these analyses in normotensive and hypertensive persons.

Uniform liquid-chromatographic conditions were developed such that we could quantify norepinephrine, epinephrine, normetanephrine, metanephrine, vanillylmandelic acid, and 5-hydroxyindoleacetic acid in urine by using a single mobile phase of monochloroacetic acid and citric acid, 0.1 mol/L each. All compounds were separated on a C18 column and detected electrochemically at a potential of +0.800 V. Optimization of these uniform chromatographic conditions significantly shortens the changeover time required from one assay to another, resulting in a substantial savings of time and cost to the laboratory.

We describe a convenient and specific radioimmunoassay for urinary metanephrine, in which we used an antiserum generated in rabbits by injecting with synephrine conjugated to bovine serum albumin as described by Grota and Brown [Endocrinology 98, 615 (1976)]. The antiserum is specific for metanephrine and epinephrine. Epinephrine cross reaction is minimized to 1% by adding 2 ng of epinephrine to each assay tube. Incubation time is 12 h. The sensitivity of the assay is 40 pg. Within-and between-assay coefficients of variation are 7.0 and 9.7%, respectively. We assayed first-voided morning urine and 24-h urine specimens from 15 normal subjects before and after acid hydrolysis, to assess total and free metanephrine concentrations. Mean (+/-SD) total and free urinary metanephrine excretion were 17 +/- 11 and 3.5 +/- 1.0 micrograms/24 h in these subjects; corresponding values for the morning specimens were 13.4 +/- 8.3 and 3.5 +/- 1.5 micrograms/liter, respectively.

A radioimmunoassay involving an 125I ligand has been developed and applied to the measurement of urinary metanephrine. To validate the clinical usefulness of this assay, we compared measurement of metanephrine by radioimmunoassay and of total urinary metanephrines by the Pisano colorimetric method. The radioimmunoassay is specific for metanephrine, whereas the colorimetric method measures both metanephrine and normetanephrine. We used both methods to determine urinary metanephrine or total metanephrines in subjects with essential hypertension, pheochromocytoma, the syndrome of multiple endocrine adenomatosis type 2, and normotensive volunteers. The mean and upper limit of normal (3 SD) for metanephrine by radioimmunoassay in our normotensive volunteers was 94.2 microgram/24 h and 229 microgram/24 h, respectively, which compares well with reported values of 87.6 microgram/24 h and 319 microgram/24 h by non-radioimmunoassay methods. Both radioimmunoassay and colorimetry accurately identified five patients with known pheochromocytoma. Good correlation (r = 0.993) was demonstrated between the two assays in a comparison of patients with essential hypertension and pheochromocytoma. We conclude that the radioimmunoassay is at least equivalent to the colorimetric methods in distinguishing pathological and normal catecholamine secretion, and is faster, more precise, and 1000-fold more sensitive.

We describe a sensitive procedure for measuring the basic catecholamine metabolites--normetanephrine, metanephrine, and 3-methoxytyramine--in urine by use of liquid chromatography, with electrochemical detection. These substances are isolated from hydrolyzed urine by passage through small ion-exchange columns and then pre-concentrated by a rapid set of solvent extractions before being injected onto the liquid-chromatographic column. Concentration and instrumental response are linearly related to 2.0 mg/liter for all three compounds, and practical lower detection limits are about 20 micrograms/liter for actual urine samples. The high selectivity that accrues from the combination of the extraction procedure, high-performance liquid chromatography, and electrochemical detection makes this procedure suitable for quantitative studies of catecholamine metabolism. A set of six samples can be taken through the extraction procedure in parallel in less than 2 h; once prepared, the extracts can be analyzed at a rate of three per hour.

A procedure has been developed for determining the O-methylated catecholamine metabolites, normetanephrine and metanephrine, in urine by use of radial-compression liquid chromatography followed by electrochemical detection. Normetanephrine and metanephrine are isolated from hydrolyzed urine by ion-exchange on small, commercially available, disposable columns and preconcentrated by solvent extraction. They are then separated by reversed-phase ion-pair chromatography, with use of a radial compression cartridge and radial compression module, and quantified with 3-methoxy-4-hydroxybenzylamine as internal standard. Normetanephrine, metanephrine, and the internal standard are separated from interfering peaks in about 15 min. The method is applicable to the relatively low amounts of normetanephrine (100-600 micrograms/24 h) and metanephrine (50-400 micrograms/24 h) found in normal subjects and patients with depressive disorders or hypertension. Within-day CVs ranged from 1.1 to 2.2% for normetanephrine and 1.2 to 6.9% for metanephrine; the corresponding between-day CVs were 4.9 and 5.7% over these ranges.

In this method for measuring metanephrine and normetanephrine in urine, they are freed from their conjugates by hydrolysis in acid, neutralized, and isolated on a cation-exchange resin. Both metanephrines are co-eluted with ammoniacal methanol and the eluate is concentrated 10-fold with respect to the original urine volume by evaporation and reconstitution. The recovered metanephrines ae separated by reversed-phase (C18) "high-pressure" liquid chromatography with use of a mobile phase consisting of 10 mmol/L trichloroacetate, pH 2.7, containing 30 mL of acetonitrile per liter. The metanephrines are detected at 365 nm after being converted to vanillin in a post-column reaction with alkaline periodate. Total metanephrine values for most patients were appreciably lower than values obtained by the comparison procedure, that of Pisano (Clin. Chim. Acta 5: 406, 1970); however, results obtained by the two methods for two patients with pheochromocytomas agreed well.

We describe a simplified fluorometric method for quantitatively determining metanephrines in urine. The method is based on the elution of metanephrines from an ion-exchange column followed by production of a fluorescent derivative that has excitation and emission maxima of 405 nm and 505 nm, respectively. Metanephrine concentrations as great as 7.0 mg/L were within the linear range of the assay. Analytical recovery of metanephrines was 82-87%. Cross contamination by catecholamines was insignificant. The upper range of normal was 520 micrograms/24 h. Within-run and between-run CV's were 5% and about 9%, respectively. Twenty-four-hour urine specimens from five patients with pheochromocytoma showed above-normal amounts of the metanephrines by this method.

Measurements of urinary metanephrines by the Pisano procedure (Clin. Chim. Acta 5: 406, 1960) are unreliable in patients who are taking propranolol owing to the presence of 4-hydroxypropranolol in the urine. Three properties of this propranolol metabolite lead to interference: (a) it is absorbed and eluted from ion-exchange resins under the conditions Pisano used for metanephrine isolation, (b) at high pH it absorbs at 350 to 360 nm, and (c) it is oxidized by periodate to a substance with negligible absorption in this region. Because 350 to 360 nm is the wavelength used to quantitate vanillin, the product formed from periodate oxidation of metanephrines, and because the unoxidized eluate is used as a specimen blank, the presence of 4-hydroxypropranolol spuriously decreases the measured urinary metanephrines; this has special significance because patients being tested for increased metanephrines are also likely to be receiving propranolol for hypertension.

We studied the recently developed Abbott fluorescence polarization immunoassay (FPIA) for amphetamine and methamphetamine in urine and compared the results with those of the Syva enzyme-multiplied immunoassay technique (EMIT) and a gas-chromatographic assay. The FPIA method showed a limit of quantification of 0.3 mg/L, comparable with the lower cutoff of the EMIT assay. FPIA demonstrated greater specificity than the EMIT assay: phenylpropanolamine and ephedrine showed extremely limited cross reactivity with the FPIA antibody. Analysis of 249 urine specimens by all three methods clearly demonstrated the FPIA method to be acceptable for screening for amphetamine and methamphetamine in urine.