Anal. Chem. 2006, 78, 6628-6633
Development and Evaluation of a Candidate Reference Measurement Procedure for the Determination of Progesterone in Human Serum Using Isotope-Dilution Liquid Chromatography/ Tandem Mass Spectrometry Susan S.-C. Tai,* Bei Xu,† and Michael J. Welch
Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8392
Progesterone is a steroid hormone that is involved in regulating female reproductive processes. Its concentration in blood is measured to determine ovarian function. A candidate reference measurement procedure for progesterone in human serum involving isotope dilution coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The progesterone along with its internal standard (progesterone-13C2) was extracted from the serum matrix using liquid-liquid extraction prior to reversed-phase LC/MS/ MS. The accuracy of the measurement was evaluated by a comparison of results of this method on a lyophilized human serum reference material for progesterone [Certified Reference Material (CRM) 347] with the certified values determined by gas chromatography/mass spectrometry (GC/MS) reference methods and by a recovery study for the added progesterone. The results of this method for progesterone agreed with the certified value within the uncertainty of the measurements for the CRM 347. The recovery of the added progesterone ranged from 100.1 to 100.9%. This method was applied to the determination of progesterone in frozen serum samples from three individual female donors with the progesterone concentrations ranging from 0.151 to 24.42 ng/g. Excellent reproducibility was obtained with within-set coefficients of variation (CVs) ranging from 0.1 to 1.4%, and between-set CVs ranging from 0.3 to 0.5%. Excellent linearity was also obtained with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.9998 to 1.0000. The detection limit at a signal-to-noise ratio of ∼3 was 1.8 pg of progesterone. This well-characterized LC/MS/MS method for serum progesterone, which demonstrates good accuracy and precision, low susceptibility to interferences, and comparability with GC/MS reference methods, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for progesterone can be compared and that will 6628 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
serve as a standard of higher order for measurement traceability. Progesterone is a steroid hormone that is involved in regulating female reproductive processes. Progesterone participates in the regulation of the menstrual cycle and is especially important in preparing the uterus for the implantation of the blastocyst and in the maintenance of pregnancy. In nonpregnant women, progesterone is secreted mainly by the corpus luteum in the ovary formed by an ovarian follicle following the discharge of its ovum. During pregnancy, the placenta becomes the major source of this hormone. Serum progesterone concentrations in adult females normally range from ∼0.15 to ∼25 ng/mL but can rise to ∼230 ng/mL during pregnancy.1 Progesterone is largely bound to protein in circulation. Its concentration in blood is measured to determine ovarian function. Serum progesterone measurements are routinely performed using methods based upon immunoassay, an approach with high sensitivity, but which is prone to nonspecificity for many analytes. There is a need for critically evaluated reference measurement procedures (RMPs) for serum progesterone. RMPs can be used to directly assess the accuracy of routine methods or can be used to assign or verify the concentrations of reference materials, as well as controls and calibrators used in routine methods. They also provide a means for demonstrating traceability of routine methods and materials to high-order reference materials. Recently, the International Organization for Standardization (ISO) published ISO 15193 (In vitro diagnostic systems-Measurement of quantities in samples of biological origin-Presentation of reference measurement procedures)2 that describes the requirements of an RMP for clinical diagnostic markers. The Joint Committee for Traceability in Laboratory Medicine (JCTLM)3 is reviewing potential RMPs and compiling a list of those that meet the requirements of ISO 15193. At present, there are two RMPs for * To whom correspondence should be addressed. E-mail:
[email protected]. † Permanent address: National Research Center for Certified Reference Materials, Beijing 100013, China. (1) Tietz, N. W. In Tietz Textbook of Clinical Chemistry, 2nd ed.; Burtis, C. A., Ashwood, E. R., Eds.; Saunders: Philadelphia, 1994; pp 1865-1866. (2) ISO 15193. 2003; http://www.iso.ch/iso/en/CatalogueListPage. CatalogueList. (3) JCTLM. 2005; http://www.bipm.org/en/committees/jc/jctlm/jctlm-db. 10.1021/ac060936b Not subject to U.S. Copyright. Publ. 2006 Am. Chem. Soc.
Published on Web 07/29/2006
Figure 1. Structures of progesterone and progesterone-13C2 (*, 13C labeling).
serum progesterone approved by the JCTLM.4-7 Both methods involve liquid/liquid extraction to isolate progesterone from the serum and then further purification of progesterone from the serum extract by the use of cyclodextrins6 or Lipidex 5000 chromatography4 and derivatization of progesterone prior to gas chromatography/mass spectrometry (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.8-14 LC/MS-based methods offer a powerful tool for the determination of nonvolatile and thermally labile compounds, usually without the need for derivatization. Sample preparation is less extensive with LC/MS-based methods. At present, there are no RMPs using LC/MS-based methods for measurement of serum progesterone approved by the JCTLM. Recently, the National Institute of Standards and Technology (NIST) developed an isotope-dilution method coupled with LC/ MS/MS for serum progesterone. To the best of our knowledge, this method is the first reported LC/MS/MS method for the measurement of progesterone in serum. This method measures total progesterone in serum, both free and protein-bound forms. The method involves liquid-liquid extraction to isolate progesterone from serum prior to reversed-phase LC/MS/MS analysis. This method was found to be free from interferences by testing the structure analogues of progesterone under the same assay conditions used for progesterone measurement. The accuracy of the measurement was evaluated by a comparison of results of this RMP on Certified Reference Material (CRM) 3475,15 [from the European Commission Institute for Reference Materials and Measurements (IRMM), formerly Community Bureau of Refer(4) Siekmann, L. J. Steroid Biochem. 1979, 11, 117-123. (5) Thienpont, L.; Siekmann, L.; Lawson, A.; Colinet, E.; De, L. A. Clin. Chem. 1991, 37, 540-546. (6) Thienpont, L. M.; De, B., VI; Stockl, D.; De Leenheer, A. P. Anal. Chem. 1994, 66, 4116-4119. (7) Thienpont, L. M.; Van, N. B.; Stockl, D.; Reinauer, H.; De Leenheer, A. P. Eur. J. Clin. Chem. Clin. Biochem. 1996, 34, 853-860. (8) Tai, S. S.; Sniegoski, L. T.; Welch, M. J. Clin. Chem. 2002, 48, 637-642. (9) Tai, S. S.; Bunk, D. M.; White, E. V.; Welch, M. J. Anal. Chem. 2004, 76, 5092-5096. (10) Tai, S. S.; Welch, M. J. Anal. Chem. 2004, 76, 1008-1014. (11) Tai, S. S.; Welch, M. J. Anal. Chem. 2005, 77, 6359-6363. (12) Thienpont, L. M.; Fierens, C.; De Leenheer, A. P.; Przywara, L. Rapid Commun. Mass Spectrom. 1999, 13, 1924-1931. (13) De, B., VI.; Hou, P.; Stockl, D.; Thienpont, L. M.; De Leenheer, A. P. Rapid Commun. Mass Spectrom. 1998, 12, 1099-1103. (14) Tai, S. S.; Xu, B.; Sniegoski, L. T.; Welch, M. J. Anal. Chem. 2006, 78, 3393-3398.
ence (BCR)] with the certified value determined by GC/MS reference methods and by a recovery study for the added progesterone. The reproducibility of this method was evaluated by repeated measurements of frozen serum samples from three individual female donors. 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. Progesterone is light sensitive. Samples and standards containing progesterone were handled with minimal exposure to light (incandescent light at reduced intensity were used). The progesterone reference compound used for this work was obtained from Sigma (St. Louis, MO). The impurities in this progesterone material were evaluated at NIST by liquid chromatography/ultraviolet, differential scanning calorimetry (DSC), and gas chromatography-flame ionization detection (GC-FID). Moisture content was measured at NIST by Karl Fischer titration. Appropriate corrections were made for impurities and moisture. A carbon-13-labeled internal standard, progesterone-13C2 (3,4-13C2), with an isotopic purity of 90% was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). A Zorbax Eclipse XDBC18 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. Frozen human serum samples from individual female donors were obtained from Interstate Blood Bank, Inc. (Memphis, TN). Samples of Seracon II, defibrinated, dialyzed, delipidated, and charcoal-stripped frozen human serum were obtained from Seralogicals Corp. (Norcross, GA). The following compounds were used for interference testing: 9(11)5β-prognen-3,20-dione, 9(11),16-(5R)-prognadien-3β-ol-20-one, 5-pregnen-3,20-dione,5,16-pregnadien-3β-ol-20-one, 5β-pregnan-3,20-dione(5β-dihydroprogesterone),and5R-pregnan-3,20-dione(allopregnanedione), all obtained from Steraloids Inc. (Newport, RI). Structures of progesterone and progesterone-13C2 are shown in Figure 1. Preparation of Calibrators. Two independently weighed standard stock solutions of progesterone were prepared. Approximately 2 mg of the progesterone reference compound for each stock solution was accurately weighed on an analytical balance and dissolved in anhydrous ethanol in a 250-mL volumetric flask. A working solution was prepared from each stock by diluting 2.0 mL in anhydrous ethanol in a 250-mL volumetric flask. The concentrations of progesterone in the working standard solutions were ∼60 pg/µL. A solution of isotopically labeled internal standard, progesterone-13C2, at a concentration of 53.45 pg/µL (progesterone-13C2 solution 1) was prepared in the same way as the unlabeled progesterone. A more diluted internal standard solution (progesterone-13C2 solution 2) at a concentration of 5.345 pg/µL was prepared by diluting 20.0 mL of progesterone-13C2 solution 1 in anhydrous ethanol in a 200-mL volumetric flask. Three aliquots from each working standard solution of progesterone were spiked with progesterone-13C2, yielding six standards (15) Thienpont, L. M.; De Leenheer, A. P.; Siekmann, L.; Colinet, E. S. EUR 12282 EN 1989.
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with the mass ratios of unlabeled to labeled compound ranging from 0.5 to 1.5. The mixtures (283-494 µL) were dried under nitrogen at 45 °C and reconstituted with 300 µL of methanol containing 0.5 mL/L acetic acid for LC/MS/MS analysis. The progesterone concentration range of the calibration curve was ∼18-54 pg/µL (for mass ratio range of 0.5-1.5). Sample Preparation. Commercially available frozen serum samples from three individual female donors (0.15, 3.45, and 24.42 ng/g for concentrations 1, 2, and 3, respectively) were used for this study. Samples were prepared in three different sets, each set consisting of triplicate aliquots from each of the three concentrations of progesterone. Each aliquot (4, 2, and 1 g for concentrations 1, 2, and 3, respectively) was accurately weighed into a 50-mL glass centrifuge tube and spiked with an appropriate amount of progesterone-13C2 (the progesterone-13C2 solution 1 was used for concentrations 2 and 3, and the progesterone-13C2 solution 2 was used for concentration 1) to get an ∼1:1 ratio of analyte to internal standard. After equilibration at room temperature for 1 h, the pH of each sample was adjusted to pH 9.8 ( 0.2 with 0.1 g/mL carbonate buffer, pH 9.8 (800, 400, and 200 µL for concentrations 1, 2, and 3, respectively). The progesterone was then extracted from the serum matrix with hexane (16 mL for concentration 1, and 8 mL for concentrations 2 and 3). Each sample was shaken vigorously for 10 min using a mechanical shaker to allow complete mixing. The upper hexane layer was transferred to another 50-mL centrifuge tube. Hexane extraction was repeated once more with 8 mL of hexane (shaken for 3 min). The combined hexane extract was dried under nitrogen at 45 °C, and the residue was reconstituted with methanol containing 0.5 mL/L acetic acid (100, 180, and 600 µL for concentrations 1, 2, and 3, respectively) for LC/MS/MS analysis. The progesterone concentrations of samples were first tested by a preliminary experiment where arbitrary amounts of the internal standard were chosen, and a wider range of mass ratios (e.g., 0.3-4.5) for the standards was used. Once the approximate progesterone concentration was determined, the quantity of internal standard was calculated to get a 1:1 ratio. Absolute Recovery of Progesterone. Stripped serum samples spiked with known amounts of progesterone were used to evaluate the recovery of progesterone from serum with this extraction method. Two groups of samples were prepared. For the first group, stripped serum was spiked with a known amount of progesterone before extraction and progesterone-13C2 after extraction. For the second group, both progesterone and progesterone13C were spiked before extraction. The samples were processed 2 as described above in the sample preparation for LC/MS/MS measurement. The absolute recovery of progesterone from serum was calculated from the comparison of the intensity ratios of the two groups. Equilibration. Commercially available frozen human serum samples from a single donor were used for this study. The concentration of progesterone in this serum material was 2.57 ng/ g. Twelve 2-g aliquots were taken for the equilibration study. An appropriate amount of progesterone-13C2 was added to each aliquot, and triplicate aliquots for each of the four time intervals (0.5, 1, 2, and 3 h) were equilibrated at room temperature. The samples were processed as described above for LC/MS/MS measurement. 6630
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Recovery of the Added Progesterone (Accuracy Test). Commercially available frozen human serum samples from a single donor were used for this study. Three 4-g aliquots for samples without unlabeled progesterone added and nine aliquots (three 2-g aliquots for each of concentrations 1 and 2, and three 1-g aliquots for concentration 3) for samples with unlabeled progesterone added were taken for a study of the accuracy of the method. A known amount of unlabeled progesterone was added to the nine aliquots, three each with 2.146, 8.880, and 24.089 ng/g of progesterone. No progesterone was added to the three 4-g aliquots. An appropriate amount of progesterone-13C2 was added to each aliquot to get an ∼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 (3-10 µL) of standards or sample extracts were separated by LC with a gradient mobile phase consisting of 0.5 mL/L acetic acid in wateracetonitrile. The gradient was initially set at water-acetonitrile (56:44 by volume) for 28 min, ramped to 100% acetonitrile at 28.1 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 315.2 f 97.1 and m/z 317.1 f 99.1 were monitored for progesterone and progesterone-13C2, respectively. The dwell times were 0.25 s for MRM. The curtain gas and collision gas were nitrogen at settings of 242 (35 psi) and 35 kPa (5 psi), respectively. The ion source gas 1 and ion source gas 2 were air, both at settings of 483 kPa (70 psi). The electrospray voltage was set at 5500 V, and the turbo gas temperature was maintained at 600 °C. The declustering potential, entrance potential, collision energy, and collision exit potential were set at 72, 10, 33, and 6 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 progesterone and other structural analogues of progesterone having relative molecular masses close to those of progesterone and progesterone13C were tested as potential interferences using the LC/MS/MS 2 method described above for progesterone measurement. Statistical Treatment. Statistical treatment of the data was in accord with NIST guidelines,16 which conform with the ISO Guide to the Expression of Uncertainty in Measurement.17 Potential sources of uncertainty were evaluated and those factors that could (16) Taylor, B. N.; Kuyatt, C. E. NIST Technical Note 1297, 1994 (available at http://physics.nist.gov/Pubs/guidelines/contents.html). (17) ISO. Guide to the Expression of Uncertainty in Measurement; International Organization for Standardization: Geneva, Switzerland, 1993.
Figure 2. Selected ion chromatograms by LC/MS/MS for progesterone and progesterone-13C2 from a serum sample at progesterone concentration of 3.45 ng/g.
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 weighing of the reference compound, and on an allowance for unknown systematic errors in the sample preparation. 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. Hexane was used to remove progesterone from endogenous binding proteins, and progesterone was then extracted into hexane from the serum matrix. This method measures total progesterone in serum, both free and proteinbound forms. The liquid-liquid extraction of serum produced a clean extract with no interference detected at ions monitored for progesterone by LC/MS/MS. The absolute recovery of progesterone from the serum with this extraction method averaged 89% (1.4% CV, n ) 6). With the isotope dilution, relative recoveries of the progest-
Table 1. Recovery of Added Progesteronea
conc
added, ng/g
expected, ng/g
mean detected, ng/g
mean recovery, %
SD %, n)3
CV %, n)3
1 2 3
0 2.146 8.880 24.089
nab 2.255 8.989 24.198
0.109 2.265 9.072 24.224
nab 100.5 100.9 100.1
nab 1.0 0.3 0.4
0.2 1.0 0.3 0.4
a
Based on known additions to a serum sample. b na, not applicable.
erone and the progesterone-13C2 internal standard should be equal if equilibration is achieved. Therefore, the absolute recoveries are not critical, since it is the ratio of unlabeled to labeled progesterone that is measured. (2) Equilibration. Complete equilibration of progesterone in serum with the spiked internal standard is necessary for accurate measurement. The time required for progesterone to be equilibrated with the internal standard, progesterone-13C2, was investigated. If the measured ratios exhibit a systematic trend over the periods tested, equilibration has not been achieved. No such trend was observed; the results from 0.5 h were indistinguishable from those at the longer times. Therefore, it appears that equilibration between the labeled and unlabeled forms is complete by 0.5 h. For convenience, the time chosen was 1 h. (3) Recovery of the Added Progesterone. The recoveries of the added progesterone are listed in Table 1. The average concentration of endogenous progesterone in the serum material tested was 0.109 ng/g. This material was spiked with progesterone at three different concentrations. The amounts of progesterone recovered and added were in very good agreement for all three Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
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concentrations; the mean recoveries ( standard deviations were 100.5 ( 1.0, 100.9 ( 0.3, and 100.1 ( 0.4% for samples with added progesterone of 2.146, 8.880, and 24.089 ng/g, respectively. (4) Interference Testing. If a structural analogue of progesterone were to coelute with progesterone during the chromatographic separation, it could bias the results. The metabolites or structural analogues of progesterone having molecular masses close to that of progesterone were tested to determine whether they could interfere with the measurement of progesterone. The structural analogues (having relative molecular masses of 314 and 316) listed in the Materials section were completely separated from progesterone under the LC conditions used for progesterone measurement. (5) Comparison to Certified Value of CRM 347. As another accuracy assessment for the RMP, samples of CRM 347 were analyzed in triplicate as described in the Experimental Section and results were compared to the certified value determined using GC/MS reference methods. The LC/MS/MS results of mass fractions of progesterone in serum (ng/g) were converted to mass concentrations (ng/mL) by multiplying by the density of the serum. The mean concentration of progesterone in CRM 347 by our method (3.139 ( 0.069 ng/mL; mean ( expanded uncertainty) agreed with the certified value (3.185 ( 0.065 ng/mL; mean ( expanded uncertainty) within the uncertainty of the measurements for this CRM, demonstrating comparability of our LC/MS/MS method with GC/MS reference methods. Measurement of Serum Materials. From a preliminary measurement, it was found that the calibration curve was linear with a correlation coefficient of 1.0000 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 ∼1 and a much shorter ratio range for the calibration standards (0.5-1.5) was used. This method was applied to frozen serum samples from three individual female donors with the progesterone concentrations ranging from 0.151 to 24.42 ng/g (the normal serum progesterone range for adult females). Samples were prepared and analyzed in three different sets, each set consisting of three concentrations of progesterone. The results are shown in Table 2. Excellent reproducibility was obtained for all three concentrations with within-set CVs ranging from 0.1 to 1.4%, and between-set CVs ranging from 0.3 to 0.5%. Excellent linearity was obtained with correlation coefficients (R) of all linear regression lines ranging from 0.9998 to 1.0000. A typical regression line was y ) 1.0992x + 0.0150 (R ) 0.9999; standard error, 0.0062; n ) 12). The linear calibration curves allowed the use of simple linear regression for calculating the weight ratios of the samples. The detection limit at a signal-to-noise ratio of ∼3 was 1.8 pg. Selected ion chromatograms for progesterone and progesterone-13C2 are shown in Figure 2. Statistical Analysis of Results. A summary of the statistical analysis for the results is shown in Table 3. The two measurements of each sample were averaged to obtain a sample mean. For each level, three sets of three samples were analyzed. An analysis of variance found that the p-values were greater than 0.05 for all the levels, indicating that set-to-set differences were not statistically 6632 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
Table 2. Reproducibility of LC/MS/MS Measurements of Progesterone in Serum overall SD,a
serum
set
mean, ng/g
ng/g
CV, %
mean, ng/g
SD,b, ng/g
CV, %
conc 1
1 2 3
0.1516 0.1520 0.1498
0.0001 0.0021 0.0003
0.1 1.4 0.2
0.1511
0.0005
0.3
conc 2
1 2 3
3.452 3.462 3.444
0.005 0.023 0.013
0.1 0.7 0.4
3.453
0.016
0.5
conc 3
1 2 3
24.362 24.484 24.415
0.054 0.056 0.041
0.2 0.2 0.2
24.421
0.069
0.3
a SD of a single measurement within a set. b SD of the mean for that level.
Table 3. Estimation of Expanded Uncertainties for LC/MS/MS Measurements of Progesterone in Serum conc 1
conc 2
conc 3
mean, ng/g
0.1511
3.4526
24.421
type A SD, ng/g SD of mean, ng/g
0.0015 0.0005
0.0156 0.0052
0.0668 0.0229
0.0015
0.0345
0.2442
0.0005
0.0104
0.0733
0.1% uncertainty of weighing, ng/g uncertainty of other systematic error, ng/g
0.0002 0.0030
0.0035 0.0030
0.0244 0.0030
combined SD uncertainty (uc), ng/g
0.0034
0.0367
0.2572
k factor expanded uncertainty (U), ng/g
2 0.0069
2 0.0734
2 0.5143
relative expanded uncertaintya, %
4.5
2.1
2.1
type B 1% uncertainty of volumetric error, ng/g 0.3% uncertainty of purity of reference compound, ng/g
a
Uncertainty of 95% confidence interval.
significant for any of the levels. Thus, the standard deviation (SD) of the mean for each level was calculated by dividing the standard 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 include uncertainties related to the use of volumetric measurements, the purity of the unlabeled progesterone reference compound, the weighing of the reference compound, and unknown systematic errors in the sample preparation (including uncertainty in the equilibration step and possible interferences from other steroids). The uncertainty of volumetric measurements was estimated to correspond to a standard deviation of 1%. The uncertainties in the purity of the reference compound and in the weighing were estimated to correspond to a standard deviation of 0.3 and 0.1%, respectively. Finally, uncertainty of other unknown sources in the sample preparation was estimated to correspond to a standard deviation of 2% of level 1 (0.003 ng/g) for all three levels. The type B contributions were much larger than the type A measure-
ment uncertainty for all levels. The relative expanded uncertainties for levels 2 and 3 were ∼2% and that for level 1 was ∼5%. CONCLUSIONS This first reported LC/MS/MS method for serum progesterone 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 progesterone can be compared. ACKNOWLEDGMENT We thank Michele Schantz (NIST) for GC-FID analysis, Barbara Porter (NIST) for DSC analysis, and Yadu Tewari (NIST) for Karl Fischer analysis for the purity assessment of the
progesterone reference compound. We thank Lorna Sniegoski (NIST) for density measurement for the serum samples. Disclaimer: 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 May 19, 2006. Accepted June 27, 2006. AC060936B
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