Anal. Chem. 2006, 78, 3393-3398
Development and Evaluation of a Candidate Reference Measurement Procedure for the Determination of 19-Norandrosterone in Human Urine Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry Susan S.-C. Tai,* Bei Xu,† Lorna T. Sniegoski, and Michael J. Welch
Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8392
19-Norandrosterone (19-NA) is the major metabolite of the steroid nandrolone, one of the most commonly abused anabolic androgenic agents. 19-NA exists mainly as the glucuronide form in human urine. A candidate reference measurement procedure for 19-NA in urine involving isotope dilution coupled with liquid chromatography/ tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The 19-NA glucuronide was enzymatically hydrolyzed, and the 19-NA along with its internal standard (deuterated 19-NA) was extracted from urine using liquid-liquid extraction prior to reversedphase LC/MS/MS. The accuracy of the measurement of 19-NA was evaluated by a recovery study of added 19NA. The recovery of the added 19-NA ranged from 99.1 to 101.4%. This method was applied to the determination of 19-NA in urine samples fortified with 19-NA glucuronide at three different concentrations (equivalent to 1, 2, and 10 ng/mL 19-NA). Excellent reproducibility was obtained with within-set coefficients of variation (CVs) ranging from 0.2 to 1.2%, and between-set CVs ranging from 0.1 to 0.5%. Excellent linearity was also obtained with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.9997 to 0.9999. The detection limit for 19-NA at a signal-to-noise ratio of ∼3 was 16 pg. The mean results of 19-NA yielded from hydrolysis of 19-NA glucuronide compared well with the theoretical values (calculated from the conversion of 19-NA glucuronide to 19-NA) with absolute relative differences ranging from 0.2 to 1.4%. This candidate reference measurement procedure for 19NA in urine, which demonstrates good accuracy and precision and low susceptibility to interferences, can be used to provide an accuracy base to which routine methods for 19-NA can be compared and that will serve as a standard of higher order for measurement traceability. 19-Norandrosterone (19-NA) is the major metabolite of the steroid nandrolone (19-nortestosterone),1 one of the most com* To whom correspondence should be addressed. E-mail:
[email protected]. † Permanent address: National Research Center for Certified Reference Materials, Beijing 100013, China. 10.1021/ac052237p Not subject to U.S. Copyright. Publ. 2006 Am. Chem. Soc.
Published on Web 04/08/2006
monly abused anabolic androgenic agents by athletes. Nandrolone, a synthetic steroid, promotes muscle growth similar to that of the endogenous male hormone, testosterone. The International Olympic Committee has prohibited the use of this drug since 1976. The World Anti-Doping Agency (WADA) statistics for 2004 show that 36% of all reported adverse analytical findings from their WADA-accredited sports testing laboratories were for anabolic agents. Of the positive cases of anabolic agent abuse, 28% were for the steroid nandrolone;2 thus, this steroid represents one of the most commonly used banned drugs. In the body, 19-NA can exist as the glucuronide form, as the sulfate form, or as the free steroid, with the glucuronide being the major metabolite in human urine. Trace amounts of endogenous 19-NA have been reported in human urine.3-6 Both glucuronide and sulfate forms of endogenous 19-NA were detected in urine, whereas only glucuronide was detected when exogenous nandrolone was administered.6 The threshold for 19-NA as established by WADA is 2 ng/mL for detecting doping with exogenous steroid nandrolone.7 The measurand for antidoping measurements as defined by WADA is the total of free 19-NA and its glucuronide expressed as a mass concentration of 19-NA in urine.7 Various gas chromatography/mass spectrometry (GC/MS) methods have been published for the measurement of 19-NA in urine.1,4,5,8-10 These methods involve extensive sample cleanup, (1) Masse, R.; Laliberte, C.; Tremblay, L.; Dugal, R. Biomed. Mass Spectrom. 1985, 12, 115-121. (2) World Anti-Doping Agency. 2004 Adverse Analytical Findings Reported by Accredited Laboratories http://www.wada-ama.org/rtecontent/document/ LABSTATS_2004.pdf 2004. (3) Van Eenoo, P.; Delbeke, F. T.; De Jong, F. H.; De Backer, P. J. Steroid Biochem. Mol. Biol. 2001, 78, 351-357. (4) Dehennin, L.; Bonnaire, Y.; Plou, P. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 721, 301-307. (5) Hemmersbach, P.; Hagensen Jetne, A. H.; Lund, H. S. Biomed. Chromatogr. In press. (6) Le Bizec B.; Bryand, F.; Gaudin, I.; Monteau, F.; Poulain, F.; Andre, F. Steroids 2002, 67, 105-110. (7) World Anti-Doping Agency. WADA Technical Document - TD2004NA. http://www.wada-ama.org/rtecontent/document/nandrolone_aug_04.pdf 2004. (8) Le Bizec B.; Gaudin, I.; Monteau, F.; Andre, F.; Impens, S.; De Wasch, K.; De Brabander, H. Rapid Commun. Mass Spectrom. 2000, 14, 1058-1065. (9) Schanzer, W.; Donike, M. Anal. Chim. Acta 1993, 275, 23-48. (10) Galan Martin, A. M.; Maynar Marino, J. I.; Garcia de Tiedra, M. P.; Rivero Marabe, J. J.; Caballero Loscos, M. J.; Maynar Marino, M. J. Chromatogr., B: Biomed. Sci. Appl. 2001, 761, 229-236.
Analytical Chemistry, Vol. 78, No. 10, May 15, 2006 3393
liquid-liquid extraction and solid-phase extraction, and derivatization of 19-NA prior to GC/MS analysis. Recent developments of highly sensitive liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) instrumentation with robust interfaces such as electrospray or atmospheric pressure chemical ionization make possible the development of highly reproducible, selective, and sensitive LC/MS-based methods for low concentration analytes.11-16 LC/MS-based methods offer a powerful tool for the determination of nonvolatile and thermally labile compounds, usually without the need for derivatization. Sample cleanup is usually considerably less extensive with LC/MS-based methods. Recently, the National Institute of Standards and Technology (NIST; Gaithersburg, MD) developed a method using isotope dilution coupled with LC/MS/MS for the measurement of 19-NA in urine. To the best of our knowledge, an LC/MS/MS method has been reported for the identification of 19-NA in urine.8 However, no LC/MS or LC/MS/MS methods have been published for the quantitation of 19-NA in urine. This method is the first reported LC/MS/MS method for the measurement of 19NA in urine. This method measures the total of free 19-NA and its glucuronide expressed as a mass concentration of 19-NA in urine as defined by WADA. The method involves enzymatic hydrolysis to liberate 19-NA from its glucuronide form and liquidliquid extraction to isolate 19-NA from urine prior to reversedphase LC/MS/MS analysis. The International Organization for Standardization (ISO) recently published ISO 15193 (In vitro diagnostic systemsMeasurement of quantities in samples of biological originPresentation of reference measurement procedures)17 that describes the requirements of reference measurement procedures (RMPs) for clinical analytes. The Joint Committee for Traceability in Laboratory Medicine (JCTLM)18 is reviewing potential RMPs and compiling a list of those that meet the requirements of ISO 15193. RMPs are used to directly assess the accuracy of routine methods or can be used to assign or verify the concentrations of controls and calibrators used in routine methods. They also provide a means for demonstrating traceability of routine methods and materials to higher-order reference materials. At present, there is no RMP for measurement of 19-NA approved by the JCTLM. This LC/MS/MS method for 19-NA in urine was found to be free from interferences by testing the structure analogues under the same assay conditions for 19-NA measurement. The accuracy of the measurement was evaluated by a recovery study of added 19-NA. The reproducibility of this method was evaluated by repeated measurements of urine materials fortified with 19-NA glucuronide at three different concentrations. The yields of 19NA from hydrolysis of 19-NA glucuronide and the theoretical (11) Tai, S. S.; Bunk, D. M.; White, E. V.; Welch, M. J. Anal. Chem. 2004, 76, 5092-5096. (12) Tai, S. S.; Welch, M. J. Anal. Chem. 2004, 76, 1008-1014. (13) Thienpont, L. M.; Fierens, C.; De Leenheer, A. P.; Przywara, L. Rapid Commun. Mass Spectrom. 1999, 13, 1924-1931. (14) Tai, S. S.; Sniegoski, L. T.; Welch, M. J. Clin. Chem. 2002, 48, 637-642. (15) Tai, S. S.; Welch, M. J. Anal. Chem. 2005, 77, 6359-6363. (16) De Brabandere VI.; Hou, P.; Stockl, D.; Thienpont, L. M.; De Leenheer, A. P. Rapid Commun. Mass Spectrom. 1998, 12, 1099-1103. (17) ISO 15193. 2003; http://www.iso.ch/iso/en/CatalogueListPage. CatalogueList. (18) JCTLM. 2005; http://www.bipm.org/en/committees/jc/jctlm/jctlm-db.
3394 Analytical Chemistry, Vol. 78, No. 10, May 15, 2006
Figure 1. Structures of 19-NA and nandrolone.
values calculated from the conversion of 19-NA glucuronide to 19-NA were compared. All of the requirements for an RMP recognized by the JCTLM have been met except for the validation by an interlaboratory study. The comparability of this method with other methods will be determined in an interlaboratory study. The results from this study will complete the requirements for an RMP recognized by the JCTLM. EXPERIMENTAL SECTION Materials. Certified Reference Material (CRM) D555 (19-NA) from National Measurement Institute (NMI) (Pymble, Australia) was used to prepare the calibration mixtures for the measurements. CRM D555 is certified as 94.2% pure with an expanded uncertainty of 2.0% (k factor, 2). CRM D596 (19-NA glucuronide, sodium salt) from NMI was used to fortify urine samples. CRM D596 is certified as 90.2% pure with an expanded uncertainty of 2.9% (k factor, 2). Appropriate corrections were made for impurities in these two CRMs. A stable deuterium-labeled internal standard, 19-NA-d4 (2,2,4,4-d4), with an isotopic purity of 93%, was also obtained from NMI. The enzyme β-glucuronidase from Escherichia coli K12 (EC 3.2.1.31) in a solution form was obtained from Roche Diagnostics (Indianapolis, IN). A Zorbax Eclipse XDB-C 18 column [15 cm × 2.1 mm (i.d.), 3.5-µm particle diameter] was obtained from Agilent Technologies (Palo Alto, CA). Solvents used for LC/ MS/MS measurements were HPLC grade and all other chemicals were reagent grade. Samples of Standard Reference Materials (SRMs) 1507b (11-Nor-Delta-9-Tetrahydrocannabinol-9-Carboxylic Acid in Freeze-Dried Urine) and 1511 (Multi-Drugs of Abuse in Freeze-Dried Urine) obtained from the Standard Reference Materials Program at NIST were used as the urine matrix to prepare 19-NA glucuronide fortified samples. The following compounds were used for interference testing: 5β-estran-3R-ol17-one (noretiocholanolone, 19-NE) from Cerilliant (Round Rock, TX) and 5R-estran-3β-ol-17-one (norepiandrosterone, 19-NEA), 5βestran-3β-ol-17-one (norepietiocholanolone), 4-estren-17β-ol-3-one (nandrolone), 4-estren-3β,17β-diol (3β-dihydronandrolone), and 5(10)-estran-3R,17β-diol from Steraloids Inc. (Newport, RI). Structures of 19-NA and nandrolone are shown in Figure 1. Preparation of Calibrators. Two independently weighed standard stock solutions of 19-NA were prepared. Approximately 2 mg of the 19-NA reference compound for each stock solution was accurately weighed on an analytical balance and dissolved in anhydrous ethanol in a 200-mL volumetric flask. A working solution was prepared from each stock by diluting 2.0 mL in anhydrous ethanol in a 200-mL volumetric flask. The concentrations of 19-NA in the working standard solutions were ∼100 pg/ µL. A solution of isotopically labeled internal standard, 19-NA-d4, at a concentration of 114 pg/µL was prepared in the same way as the unlabeled 19-NA. Three aliquots from each working standard solution of 19-NA were spiked with 19-NA-d4 yielding six standards with the mass
ratios of unlabeled-to-labeled compound ranging from 0.5 to 1.5. The mixtures (324-530 µL) were dried under nitrogen at 45 °C and reconstituted with 120 µL of methanol containing 1 mL/L acetic acid for LC/MS/MS analysis. The 19-NA concentration range of the calibration curve was ∼0.1-0.3 ng/µL (for mass ratio range of 0.5-1.5). Sample Preparation. Two freeze-dried urine materials (SRMs 1507b and 1511), each fortified with 19-NA glucuronide at three different concentrations, were used for this study. For each material, vials of reconstituted urine samples were combined, and nine aliquots were fortified with 19-NA glucuronide at three concentrations, three each with 1.718, 3.437, and 17.142 ng/mL 19-NA glucuronide (sodium salt) for concentrations 1, 2, and 3, respectively (equivalent to 1.001, 2.002, and 9.985 ng/mL 19-NA for concentrations 1, 2, and 3, respectively). Samples were prepared in six different sets (3 sets each for SRM 1507b and SRM 1511). Each set consisted of triplicate 8.0-mL aliquots for each of concentrations 1 and 2 and triplicate 2.0 mL for concentration 3. One 8.0-mL aliquot of urine sample without fortification was also included in each set. Each aliquot was placed into a 50mL glass centrifuge tube and an appropriate amount of 19-NA-d4 was added to give an ∼1:1 ratio of analyte to internal standard. The pH of each sample was adjusted to 6.3 ( 0.1 by adding 2 mL of 0.2 mol/L phosphate buffer (pH 6.0) and a small amount of 0.5 mol/L phosphoric acid (up to 250 µL). To each sample, 400 µL of β-glucuronidase solution was added to hydrolyze 19-NA glucuronide to free 19-NA. The mixtures were incubated at 50 °C for 1 h. Following hydrolysis, each sample was cooled to room temperature and adjusted to pH 9.8 ( 0.2 with 2 mL of 0.1 g/mL carbonate buffer (pH 9.8) and a small amount of 5 mol/L ammonium hydroxide (up to 300 µL). To each sample, 2 g of sodium chloride was added, and the 19-NA was then extracted from the urine hydrolyzate with 16 mL of hexane. Each sample was shaken vigorously for 3 min using a mechanical shaker to allow complete mixing. After centrifugation of the tube for 10 min at 2000g, the upper hexane layer was transferred to another 50mL centrifuge tube. Hexane extraction was repeated once more with another 16 mL of hexane. The combined hexane extract was dried under nitrogen at 45 °C, and the residue was reconstituted with 100 µL of methanol with 1 mL/L acetic acid to a final concentration of ∼0.2 ng/µL of 19-NA for LC/MS/MS analysis. Absolute Recovery of 19-NA. The urine samples from SRM 1507b were used for the study of absolute recovery of 19-NA with the extraction method. Two groups of samples were prepared. For the first group, urine samples were spiked with a known amount of 19-NA before extraction and 19-NA-d4 after extraction. For the second group, both 19-NA and 19-NA-d4 were spiked before extraction. The samples were processed as described above in the sample preparation for LC/MS/MS measurement. The absolute recovery of 19-NA from urine was calculated from the comparison of the intensity ratios of the two groups. Recovery of the Added 19-NA (Accuracy Test). The urine samples from SRM 1507b were used for this study. Triplicate 8.0mL aliquots for each of concentrations 1 and 2 and triplicate 2.0 mL for concentration 3 were taken for a study of the accuracy of the method. A known amount of 19-NA was added to the nine aliquots, three each with 1.000, 2.001, and 9.981 ng/mL 19-NA. An appropriate amount of 19-NA-d4 was added to each aliquot to
get an approximate 1:1 ratio of analyte to internal standard, and the aliquots were processed as described above in the sample preparation for LC/MS/MS measurement. LC/MS/MS Analysis. Analysis was performed on an Applied Biosystems API 4000 (Foster City, CA) equipped with an Agilent 1100 Series LC system (Palo Alto, CA). A Zorbax Eclipse XDBC18 column was used for the analysis. Aliquots (10 µL) of standards or sample extracts were separated by LC with a gradient mobile phase consisting of 0.5 mL/L acetic acid in water-acetonitrile. The gradient was initially set at water-acetonitrile (66:34 by volume) for 40 min, ramped to 100% acetonitrile at 40.1 min, and held for 10 min to wash the column. The flow rate was 0.25 mL/ min. The column temperature was set at 30 °C. The autosampler tray temperature was set at 10 °C. Electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM) mode were used for LC/MS/MS. The transitions at m/z 277 f m/z 259 and m/z 281 f m/z 263 were monitored for 19-NA and 19NA-d4, respectively. The dwell times were 0.25 s for MRM. The curtain gas and collision gas were nitrogen at settings of 207 (30 psi) and 21 kPa (3 psi), respectively. The ion source gas 1 and ion source gas 2 were air at settings of 518 (75 psi) and 310 kPa (45 psi), respectively. The electrospray voltage was set at 5500 V, and the turbo gas temperature was maintained at 450 °C. The declustering potential, entrance potential, collision energy, and collision exit potential were set at 61, 10, 13, and 18 V, respectively. The following measurement protocol was used for LC/MS/ MS analysis. The standards were analyzed first, followed by the samples, and then by the samples and standards in reverse order. By combining the data of standards run before and after the samples, a linear regression was calculated (y ) mx + b model), which was used to convert the measured intensity ratios of analyte to mass ratios. The mass ratios were then used along with the amounts of the internal standard added to calculate analyte concentrations. Interference Testing. The metabolites of nandrolone and other structural analogues of 19-NA having relative molecular masses close to that of 19-NA were tested for interference using the LC/MS/MS method described above for 19-NA measurement. Statistical Treatment. Statistical treatment of the data was in accord with NIST guidelines,19 which conform with the ISO Guide to the Expression of Uncertainty in Measurement.20 Potential sources of uncertainty were evaluated, and those factors that could contribute significantly were used to calculate the standard uncertainty. The type A uncertainty, calculated from the imprecision of the measurements, was combined quadratically with the type B uncertainty components to determine the standard uncertainty uc. For type A, an analysis of variance calculation was performed on the measurement data to determine whether setto-set differences were statistically significant. This analysis determined the number of independent measurements n used for calculating the measurement standard deviation of the mean. The type B factor contributions used were based on knowledge about uncertainties in the purity of the reference compound, in the accuracy of volumetric addition steps, in the completeness of the hydrolysis step, and on an allowance for unknown systematic (19) Taylor, B. N.; Kuyatt, C. E. NIST Technical Note 1297, 1994 (available at http://physics.nist.gov/Pubs/guidelines/contents.html). (20) ISO Guide to the Expression of Uncertainty in Measurement; International Organization for Standardization: Geneva, Switzerland, 1993.
Analytical Chemistry, Vol. 78, No. 10, May 15, 2006
3395
Table 1. Recovery of Added 19-NAa conc
added, ng/mL
mean detected, ng/mL
mean recovery, %
SD, %, n)3
CV, %, n)3
1 2 3
1.000 2.001 9.981
1.014 1.983 9.922
101.4 99.1 99.4
0.5 0.6 0.5
0.5 0.6 0.5
a
Based on known additions to urine samples of SRM 1507b.
errors in the LC/MS/MS analysis (including a small amount of endogenous 19-NA). For calculation of the expanded uncertainty U, uc was multiplied by the coverage factor k ) 2, according to the ISO convention. RESULTS AND DISCUSSION Evaluation of Critical Parameters. Reference measurement procedures must be thoroughly tested for sources of bias. The following sections discuss the evaluation of critical parameters that potentially could bias the results. (1) Extraction. The liquid-liquid extraction produced a clean extract with no interference detected at ions monitored for 19NA by LC/MS/MS. The absolute recovery of 19-NA from the urine with this extraction method averaged 95% (0.5% CV, n ) 3). With the isotope dilution, relative recoveries of the 19-NA and the 19-NA-d4 internal standard should be equal. Therefore, the absolute recoveries are not critical, since it is the ratio of unlabeled to labeled 19-NA that is measured. (2) Recovery of the Added 19-NA. The recoveries of the added 19-NA are listed in Table 1. No endogenous 19-NA was detected in the urine material of SRM 1507b. This material was spiked with 19-NA at three different concentrations. The amount of 19-NA recovered and added were in very good agreement for all three concentrations; the mean recoveries ( standard deviations were 101.4 ( 0.5, 99.1 ( 0.6, and 99.4 ( 0.5% for samples with added 19-NA of 1.000, 2.001, and 9.981 ng/mL, respectively. (3) Interference Testing. If a structural analogue of 19-NA were to coelute with 19-NA during the chromatographic separation, it could bias the results. The known metabolites of nandrolone, 19-NEA and 19-NE (having the same relative molecular mass as that of 19-NA, Mr ) 276),1,21-23 were tested to determine whether they could interfere with the measurement of 19-NA. These two compounds were completely separated from 19-NA with retention times of 21, 27, and 34 min for 19-NEA, 19-NE, and 19NA, respectively, under the LC conditions for 19-NA measurement. The other structural analogues (having relative molecular masses of 274 and 276) listed in the material section were also completely separated from 19-NA. Measurement of Urine Materials. From a preliminary measurement, it was found that the calibration curve was linear with a correlation coefficient (R) of 0.9999 over a wider range of mass ratios (e.g., 0.3-4.5, data not shown). However, for a reference method application, measurements should be made under conditions that produce the best achievable accuracy and precision. Therefore, the samples were spiked at a mass ratio of (21) Engel, L. L.; Alexander, J.; Wheeler, M. J. Biol. Chem. 1958, 231, 159164. (22) Schanzer, W. Clin. Chem. 1996, 42, 1001-1020. (23) Wolthers, B. G.; Kraan, G. P. J. Chromatogr., A 1999, 843, 247-274.
3396 Analytical Chemistry, Vol. 78, No. 10, May 15, 2006
Table 2. Reproducibility of LC/MS/MS Measurements of 19-NA from Urine of SRM 1507b Fortified with 19-NA Glucuronide overall SD,a ng/mL
CV, %
mean, ng/mL
SD.b ng/mL
CV, %
urine
set
mean, ng/mL
conc 1
1 2 3
1.005 1.006 0.997
0.003 0.007 0.005
0.3 0.7 0.5
1.003
0.003
0.3
conc 2
1 2 3
1.980 2.001 1.970
0.006 0.006 0.012
0.3 0.3 0.6
1.984
0.009
0.5
1 2 3
9.809 9.963 9.868
0.058 0.063 0.053
0.6 0.6 0.5
9.880
0.045
0.5
conc 3
a SD of a single measurement within a set. b SD of the mean for that level.
Table 3. Reproducibility of LC/MS/MS Measurements of 19-NA from Urine of SRM 1511 Fortified with 19-NA Glucuronide overall SD,a ng/mL
CV, %
mean, ng/mL
SD,b ng/mL
CV, %
urine
set
mean, ng/mL
conc 1
1 2 3
1.014 1.001 1.003
0.012 0.005 0.006
1.2 0.5 0.6
1.006
0.003
0.3
conc 2
1 2 3
1.985 1.993 1.991
0.006 0.009 0.009
0.3 0.4 0.5
1.990
0.003
0.2
conc 3
1 2 3
9.869 9.846 9.829
0.024 0.040 0.053
0.2 0.4 0.5
9.848
0.010
0.1
a SD of a single measurement within a set. b SD of the mean for that level.
∼1 and a much shorter ratio range for the calibration standards (0.5-1.5) was used. This method was applied to two urine materials, each fortified with 19-NA glucuronide at three different concentrations (equivalent to 1.001, 2.002, and 9.985 ng/mL 19-NA). For each material, samples were prepared and analyzed in three different sets, each set consisting of the three concentrations of 19-NA glucuronide. The results are shown in Tables 2 and 3 for the materials of SRM 1507b and SRM 1511, respectively. Excellent reproducibility was obtained for both materials with within-set CVs ranging from 0.2 to 1.2% and between-set CVs ranging from 0.1 to 0.5% for all three concentrations. Excellent linearity was obtained with R of all linear regression lines ranging from 0.9997 to 0.9999. A typical regression line was as follows: y ) 1.0670x - 0.0032 (R ) 0.9999; standard error, 0.0062; n ) 12). The detection limit for 19-NA at a signalto-noise ratio of ∼3 was 16 pg. Selected ion chromatograms for 19-NA and 19-NA-d4 are shown in Figure 2. The mean results of 19-NA yielded from hydrolysis of 19-NA glucuronide for the materials of SRM 1507b (shown in Table 2) and SRM 1511 (shown in Table 3) were summarized in Table 4. The theoretical values listed in this table were calculated by multiplying the fortified amounts of 19-NA glucuronide (1.718, 3.437, and 17.142 ng/mL 19-NA glucuronide sodium salt for
Table 5. Estimation of Expanded Uncertainties for LC/MS/MS Measurements of 19-NA from Urine of SRM 1507b Fortified with 19-NA Glucuronide
type A mean, ng/mL SD, ng/mL SD of mean, ng/mL type B 1% uncertainty of volumetric error, ng/mL 1% uncertainty of purity of reference compd, ng/mL 1.5% uncertainty of hydrolysis, ng/mL 0.1% uncertainty of weighing, ng/mL uncertainty of other systematic error, ng/mL
Figure 2. Selected ion chromatograms by LC/MS/MS for 19-NA and 19-NA-d4 from a urine sample. Table 4. Comparison of Mean Results of 19-NA Yielded from Hydrolysis of 19-NA Glucuronide with Theoretical Values
material
conc
mean result, ng/mL
theor value, ng/mL
1507b 1507b 1507b
1 2 3
1.003 1.984 9.880
1.001 2.002 9.985
+0.2 -0.9 -1.1
1511 1511 1511
1 2 3
1.006 1.990 9.848
1.001 2.002 9.985
+ 0.5 -0.6 -1.4
conc 2
conc 3
1.003 0.005 0.003
1.984 0.016 0.009
9.880 0.078 0.045
0.010 0.010
0.020 0.020
0.099 0.099
0.015 0.001 0.010
0.030 0.002 0.010
0.148 0.010 0.010
combined SD uncertainty (uc), ng/mL
0.023
0.043
0.209
k factor expanded uncertainty (U), ng/mL
2 0.046
2 0.086
2 0.418
relative expanded uncertainty,a %
4.6
4.3
4.2
a
Uncertainty of 95% confidence interval.
Table 6. Estimation of Expanded Uncertainties for LC/MS/MS Measurements of 19-NA from Urine of SRM 1511 Fortified with 19-NA Glucuronide
relative difference, %
concentrations 1, 2, and 3, respectively) by a conversion factor of 0.5825, which was obtained by dividing the relative molecular mass of 19-NA (Mr ) 276.4) by the relative molecular mass of 19-NA glucuronide sodium salt (Mr ) 474.5). The mean results of hydrolysis yield for these two materials compared well with the theoretical values with absolute relative differences ranging from 0.2 to 1.4%. Statistical Analysis of Results. The summaries of the statistical analyses for the results are shown in Tables 5 and 6 for the materials of SRM 1507b and SRM 1511, respectively. For each level of each material, three sets of three samples each were analyzed. To determine whether set-to-set differences were significant, the two measurements of each sample were averaged to obtain a sample mean, which was then normalized by dividing by the overall mean of that level. For SRM 1507b, an analysis of variance found that the p-values were less than 0.05 for all the normalized sample means, indicating that set-to-set differences were statistically significant, effectively reducing the number of independent observations to the number of sets. Thus, the standard deviation of the mean for each level was calculated by dividing the standard deviation of the set means for that level by the square root of n, where n ) 3. For SRM 1511, an analysis of variance found that the p-values were greater than 0.05 for all the normalized sample means, indicating that set-to-set differences were not statistically significant. Thus, the standard deviation of the mean for each level was calculated by dividing the standard
conc 1
conc 1
conc 2
conc 3
type A mean, ng/mL SD, ng/mL SD of mean, ng/mL
1.006 0.009 0.003
1.990 0.008 0.003
9.848 0.030 0.010
type B 1% uncertainty of volumetric error, ng/mL 1% uncertainty of purity of ref compd, ng/mL 1.5% uncertainty of hydrolysis, ng/mL 0.1% uncertainty of weighing, ng/mL uncertainty of other systematic error, ng/mL
0.010 0.010 0.015 0.001 0.010
0.020 0.020 0.030 0.002 0.010
0.098 0.098 0.148 0.010 0.010
combined SD uncertainty (uc), ng/mL
0.023
0.043
0.204
k factor expanded uncertainty (U), ng/mL
2 0.047
2 0.085
2 0.407
relative expanded uncertainty,a %
4.6
4.3
4.1
a
Uncertainty of 95% confidence interval.
deviation of the sample means for that level by the square root of n, where n ) 9. To calculate the standard uncertainty uc, the standard deviation of the mean for the measurements was combined quadratically with the type B factors, which includes uncertainties related to the use of volumetric measurements, the purity of the unlabeled 19-NA reference compound, the hydrolysis step, the weighing of the reference compound, and a small amount of systematic errors contributed from endogenous 19-NA and other unknown sources (up to 0.01 ng/mL). The first two of these type B factors were estimated to correspond to a standard deviation of 1%. The uncertainty in the hydrolysis step was estimated to correspond to a standard deviation of 1.5%. The uncertainty in the hydrolysis step was small compared to the uncertainty of the purity of the 19-NA glucuronide compound (expanded uncertainty of 2.9% with k factor of 2); thus, the uncertainty corresponding to a standard deviation of half of 2.9% (1.5%) was used as the estimated uncertainty for the hydrolysis step. Finally, there was a small Analytical Chemistry, Vol. 78, No. 10, May 15, 2006
3397
uncertainty in the weighing of the reference compound estimated to correspond to a standard deviation of 0.1%. For all levels of both materials, the type B contributions were much larger than the measurement uncertainty. The relative expanded uncertainties for all three levels were ∼4-5%. CONCLUSIONS This first reported LC/MS/MS method for 19-NA in urine demonstrates good accuracy and precision and low susceptibility to interferences. Use of this candidate reference measurement procedure can provide an accuracy base to which routine methods for 19-NA can be compared.
3398
Analytical Chemistry, Vol. 78, No. 10, May 15, 2006
ACKNOWLEDGMENT Certain commercial equipment, instruments, and materials are identified in this paper to adequately specify the experimental procedure. Such identification does not imply recommendation or endorsement by NIST nor does it imply that the equipment, instruments, or materials are necessarily the best available for the purpose.
Received for review December 18, 2005. Accepted February 1, 2006. AC052237P
