Clinical Chemistry 54: 1290-1297, 2008.
First published June 12, 2008; 10.1373/clinchem.2008.105841
(Clinical Chemistry. 2008;54:1290-1297.)
© 2008 American Association for Clinical Chemistry, Inc.
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Endocrinology and Metabolism |
State-of-the-Art of Serum Testosterone Measurement by Isotope Dilution–Liquid Chromatography– Tandem Mass Spectrometry
Linda M. Thienpont1,a,
Katleen Van Uytfanghe1,
Stuart Blincko2,
Carol S. Ramsay3,
Hui Xie3,
Robert C. Doss3,
Brian G. Keevil4,
Laura J. Owen4,
Alan L. Rockwood5,6,
Mark M. Kushnir5,
Kelly Y. Chun7,
Donald W. Chandler7,
Helen P. Field8 and
Patrick M. Sluss9
1 Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Gent University, Gent, Belgium; 2
Abbott Diagnostics, Dartford, Kent, UK; 3
Abbott Diagnostics, Abbott Park, IL; 4
Department of Biochemistry, University Hospital of South Manchester, Manchester, UK; 5
ARUP Laboratories, Salt Lake City, UT; 6
Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; 7
Esoterix Inc., Calabasas Hills, CA; 8
Department of Clinical Biochemistry, Old Medical School, Leeds General Infirmary, Leeds, UK; 9
Clinical Pathology Core Laboratory, Massachusetts General Hospital, Boston, MA.
aAddress correspondence to this author at: Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Gent University, Harelbekestraat 72, B-9000 Gent, Belgium. Fax +32-9-264.81.98; e-mail linda.thienpont{at}ugent.be.
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Abstract
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Background: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution–liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID–gas chromatography (GC)–MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine.
Methods: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs.
Results: The testosterone concentrations by ID-GC-MS were 0.2–4.4 nmol/L (women), 0.2–2.0 nmol/L (hypogonadal man), and 10.1–31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90–1.11, –0.055–0.013 nmol/L, and 0.993–0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between –9.6 and 0.4%.
Conclusions: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.
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Introduction
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The measurement of serum testosterone by current commercially available direct immunoassay is a matter of concern because of its current lack of accuracy and standardization (traceability) (1)(2)(3)(4)(5). In particular, these methods are severely limited for quantifying testosterone in specimens from women and children. Recognizing the problem, the Endocrine Society and the CDC have recently started an initiative, recommending that "in the absence of other information, direct immunoassays (those performed on whole serum or plasma) perform poorly at low testosterone concentrations (i.e., women, children, and hypogonadal men) and should be avoided" (6)(7). In response to this need, several laboratories have developed extracted-serum testosterone assays based on isotope dilution–liquid chromatography (ID-LC)1
–tandem mass spectrometry (MS/MS). This method was selected because of its inherent potential for high specificity and accuracy of measurement, which stems from the combination of multiple reaction monitoring at mass-over-charge ratios (m/z) typical for precursor-to-product ion transitions of labeled and nonlabeled analyte and internal standardization of each extracted specimen analyzed. Several of the ID-LC-MS/MS measurement procedures now in use have been published for clinical application in a routine laboratory setting (8)(9)(10)(11)(12)(13)(14)(15). Unfortunately, from the published data, it is difficult to assess the accuracy and state of standardization of these assays (16) because ID-LC-MS/MS methods are most often compared with immunoassays, but not with each other, and specimens tested do not always cover the clinically important patient populations.
Here, we present a method comparison study of 4 routinely used ID-LC-MS/MS measurement procedures with an ID–gas chromatography (GC)–MS reference measurement procedure (RMP) (17). The latter is 1 of the only 2 RMPs for serum testosterone listed in the database of the Joint Committee for Traceability in Laboratory Medicine (JCTLM) (18). The RMP has been in use for >15 years in a dedicated reference measurement laboratory (18)(19). The use of an internationally recognized RMP to perform measurements is in contrast to earlier publications, in which it was assumed that discrepancies in comparison with the immunoassays were attributable to the cross-reactivity of the immunoassays (2)(4)(13). The specimens used covered clinically important patient groups.
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Materials and Methods
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study subjects
We conducted the method comparison study with 58 serum specimens obtained from 2 sources: ProMedDx and Massachusetts General Hospital (Reproductive Endocrine Unit). The 40 specimens from ProMedDx were obtained from healthy donors, 10 men (age 22–42 years) and 30 women (age 18–56 years, with 8 subjects 
45 years) who gave written informed consent. The study received institutional review board approval. The procedure used for collection and processing of blood to obtain serum is described in detail in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue8. The 18 sera from Massachusetts General Hospital were from 5 men with normal reproductive function, 8 men with hypogonadal hypogonadism, and 5 pools of serum samples. We used 2 of these pools without modification and supplemented them with 3 different concentrations of testosterone for the 3 calibration samples. All 13 male subjects provided informed consent to participate in research study protocols that had been approved and monitored by the local Human Studies Committee (selection criteria for enrollment described in the online Data Supplement). The pools were generated from discarded clinical serum specimens selected solely based on male or female identification on the labels (approved and monitored by the Human Studies Committee at Massachusetts General Hospital). Details of the respective protocols used for blood drawing, generation of serum, processing, and storage are described in the online Data Supplement.
At least 3 aliquots of each serum were distributed on dry ice to each of the participating laboratories, who confirmed that the specimens arrived frozen. Specimens were stored at –20 °C until assays were performed.
id-ms measurement procedures
The main methodological characteristics of the ID-LC-MS/MS measurement procedures used in this study are summarized in Table 1
(8)(11)(12). At the start, it was agreed that the results of the laboratories applying ID-LC-MS/MS would be presented anonymously (coded without relationship between the codes or sequence of the cited references). All assays were performed according to the standard operating procedure established and validated in each laboratory.
Details of the ID-GC-MS RMP are described elsewhere (17)(19)(20)(21)(22) (for a summary see Table 1
). Calibration was done with testosterone/[3,4-13C2]testosterone mixtures at a 1:1 isotope ratio (±15%) prepared from 3 independent working solutions, demonstrated beforehand to not differ by >1% from each (n = 6, probability 95%) (20). Each run was monitored for accuracy and precision against acceptance limits (21) from duplicate analysis of serum specimens, bearing values assigned by the second JCTLM-listed RMP (from the Deutsche Gesellschaft für Klinische Chemie und Laboratoriumsmedizin eV) (18).
testing protocol
The study prescribed analysis of each specimen to be performed in 3 (ID-LC-MS/MS) or 4 (ID-GC-MS) replicates in separate independent runs. Independent runs were defined such that on each occasion of measurement, separate sampling, specimen purification, calibration, and internal quality control was done. The results of each of the 5 participating laboratories were collected by the study coordinator.
validation and statistical data analysis
The measurement protocol allowed assessment of the imprecision (expressed as %CV) of each of the applied measurement procedures (ID-LC-MS/MS: 3 independent runs; ID-GC-MS: 4 independent runs). We obtained the median from the ranked individual CV values, calculated for all specimens that were within the reportable range of an assay. In view of the low concentrations typically expected for females and hypogonadal males, the limit of quantification (LOQ) of each ID-LC-MS/MS measurement procedure was verified in the study. For method comparisons, results (x = ID-GC-MS; y = ID-LC-MS/MS) were analyzed by weighted Deming regression (CBstat, version 5.1) and represented in a scatter plot and %-difference plot. This was done for each ID-LC-MS/MS replicate measurement vs the mean ID-GC-MS measurement for each specimen tested. In the %-difference plot, the biologically derived total error limit of 14% was included (23).
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Results
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testosterone concentrations in the different populations
The testosterone concentrations (determined by ID-GC-MS) were 0.2–4.4 nmol/L (women), 0.2–2.0 nmol/L (men with hypogonadal hypogonadism), and 10.1–31.3 nmol/L (healthy men). The concentrations in pooled samples were 0.8 and 12.1 nmol/L in women and men, respectively. The female pool was supplemented to increase the testosterone concentration to 2.7 and 7.0 nmol/L, the male pool to 17.3 nmol/L. For conversion, 10 nmol/L testosterone = 288 ng/dL.
imprecision
The median CV of the assays (ID-GC-MS: 4 independent runs; ID-LC-MS/MS: 3 independent runs) ranged from 1.0% (ID-GC-MS) to 8.1% (assay A, Table 2
). In the concentration range >5 nmol/L, the median CVs for ID-LC-MS/MS were 2.0%, 2.9%, 4.5%, and 6.6% for assays D, C, B, and A; in the range <5 nmol/L, they were 3.9%, 6.5%, 6.7%, and 8.5% for assays D, B, C, and A. With the exception of 1 outlying value for assay B, all individual CV values were <20%. The imprecision profile (Fig. 1
) shows that the CV of the ID-GC-MS assay was nearly independent of the concentration, whereas higher values were observed for the ID-LC-MS/MS procedures at low concentrations. Some values of zero for assay C were due to reporting of results with an insufficient number of digits (note: these values were included in the calculation of the median CV). Assay A showed a relatively high imprecision over the whole range. This was due to considerable between-run effects (see also regression data in Table 3
).
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Figure 1. Imprecision profile of all assays investigated (by increasing imprecision).
The CV was calculated from triplicates (A–D) and quadruplicates (Gent) of all specimens that were within the reportable range of an assay. Note the logarithmic scale of the x axis.
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limit of quantification
LOQs ranged from 0.035 to 0.30 nmol/L (Table 1
). Three specimens (1 hypogonadal man and 2 women) contained testosterone concentrations (0.18, 0.20, and 0.27 nmol/L by ID-GC-MS) below the LOQs (0.3 nmol/L and 0.26 nmol/L, respectively) of 2 of the assays and thus could not be measured by those systems.
method comparison
The weighted Deming regression parameters in Table 3
show that the mean slope values ranged from 0.90 (A) to 1.11 (B) and the mean intercept values from –0.055 (B) to 0.013 (D) nmol/L. Assay A revealed considerable between-run variation (slopes from 0.85 to 0.94). The correlation coefficients ranged from 0.993 (A and B) to 0.997 (D). Fig. 2
shows the scatter and %-difference plots of the most representative replicate (based on the slope and error distribution) of the ID-LC-MS/MS assays vs the mean of the ID-GC-MS RMP. The scatter plots (Fig. 2
, left) reveal a low dispersion of the results around the weighted Deming regression line. The %-difference plots (Fig. 2
, right) show that 7%, 11%, 14%, and 26% of the results of assays D, C, B, and A were outside the biologically derived total error limit of 14%. For assays D, C, and A, none of the results were outside a total error of 25%, whereas assay B shows 3.4% of the results outside that total error limit. The logarithmic version of the %-difference plot (Fig. 3
) additionally revealed that assay B showed a negative bias in the lowest concentration range but a positive bias throughout the remaining range of concentrations. The median deviations of the ID-LC-MS/MS procedures from ID-GC-MS were –9.6% (A), 6.4% (B), 6.8% (C), and 0.4% (D).
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Figure 2. Left: scatter plots with line of identity (dotted) and weighted Deming regression line (solid).
Right: %-difference plots with 14% total error limit (dotted line) of the most representative replicate of each ID-LC-MS/MS assay (A1, B3, C2, D1 in Table 1
) vs the mean of the ID-GC-MS reference measurement procedure.
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Figure 3. %-difference plots with logarithmic x scale and 14% total error limit (dotted line) of the most representative replicate of each ID-LC-MS/MS assay vs the mean of the ID-GC-MS reference measurement procedure.
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Discussion
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This study compared, for the first time, 4 ID-LC-MS/MS procedures for serum testosterone to a JCTLM-listed RMP (ID-GC-MS) operated in an experienced reference laboratory. The specimens tested contained testosterone concentrations between 0.2 and 31 nmol/L, which covered clinically relevant ranges for peripheral testosterone measurement in women, hypogonadal men, and healthy men. For all ID-LC-MS/MS procedures, the data showed a quality of performance over the entire concentration range tested, in stark contrast to that reported for immunoassays, which revealed severe accuracy problems, particularly in the female testosterone concentration range (1)(2)(4)(13). It is noteworthy in this regard that substantial methodological differences existed among the ID-LC-MS/MS assays, including derivatization vs none, different organic extraction and purification protocols, and instrumentation differences. Isotope dilution and specificity of detection in the multiple reaction monitoring mode underlie this improved accuracy across ID-LC-MS/MS compared with immunoassay.
The present study identified some performance differences in the 4 ID-LC-MS/MS assays investigated, however. Assay D was excellently calibrated (median deviation from ID-GC-MS 0.4%), highly reproducible (for example, slopes from 0.97 to 1.01), and highly precise (overall CV 3.5%; average CV at approximately 0.3 nmol/L 10%). The other assays showed small to medium deviations (e.g., bias) from ID-GC-MS (median 6.4%, B; 6.8%, C; –9.6%, A). This indicates a need for improvement of the assays’ calibrations and/or precision, especially in assay A. Assay B illustrates a need for improved low-range calibration, as indicated by the consistent negative bias in that range (approximately –15%) and the negative regression intercept (–0.055 nmol/L). Although the between-run reproducibility was good for assays B, C, and D, assay A requires a better between-run calibration control (the slopes, for example, ranged from 0.85 to 0.94). The assays also had a different LOQ (Table 1
), although it should be noted that some estimated it from the functional sensitivity (CV 20%), whereas others used a more conservative approach (CV 20% and bias <20%). All these data emphasize the importance of proper assay calibration, quality control, and good laboratory practices for measurements of testosterone by ID-LC-MS/MS in clinical laboratories. The study also showed that method comparisons such as these are invaluable for helping laboratories to improve their processes. For example, after this intercomparison, laboratory A uncovered and corrected an operator error related to sample mixing that was responsible for much of the variability in the results reported here. Last, the specificity of LC-MS/MS should be emphasized, which can be enhanced by using multiple mass transitions to detect interferences in individual samples. When this method is used, a measurement is considered free of interference if the intensity ratio between quantitative and qualitative (confirming) transitions agrees with a predetermined standard within an acceptable error range (24). In methods using multiple mass transitions, only transitions of high quality (in terms of signal to noise and kind of transition) should be monitored, avoiding nonspecific MS/MS transitions, such as loss of H2O, for both quantitative and qualitative transitions. Otherwise, too many samples will be rejected owing to suspected interferences. Although specificity of methods was not directly evaluated in the present study, the agreement between the evaluated and reference methods in patient samples argues in favor of a low rate of interferences for all the methods used in this study. This was confirmed by the fact that no interferences were detected in this sample set by methods that implemented 2-transition ratio-based interference detection (see Table 1
for transitions used by each method). Other factors likely contributing to the low interference rate observed in this study were extensive sample clean-up (as used in the reference ID-GC-MS measurement procedure) and assessment of interference from other steroid hormones as part of the method validation (22).
Our study showed that the ID-LC-MS/MS procedures for measurement of testosterone in serum have sufficient analytical sensitivity to distinguish between normogonadal, hypogonadal, and female testosterone concentrations, proper specificity, even in the low concentration range, and fairly good accuracy within the biologically derived total error limit. But there is also a drawback to consider. Improvements in LC-MS/MS in terms of ease of use will lead to more laboratories developing new measurement procedures for serum testosterone. Therefore, it must be emphasized that new measurement procedures need to be thoroughly validated, including their traceability to an internationally accepted RMP performed in a competent reference laboratory. Indeed, standardization is a crucial foundation for improved patient care. Without acknowledging the importance of this, it is likely that an uncontrolled proliferation of MS measurement procedures will rapidly vitiate the current good accuracy state that our data indicate is achievable for ID-LC-MS/MS. This conclusion is true for all methods used for measurement of testosterone. Therefore, the requirement for standardization should also be extended to direct immunoassays, whose reportable ranges should be limited to concentrations in which accuracy can be documented relative to internationally accepted reference measurements.
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Acknowledgments
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Grant/Funding Support: Patient and specimen handling costs were supported, in part, by the Reproductive Endocrine Unit of the Massachusetts General Hospital. Abbott provided the funding for the reference study measurements and distribution of samples.
Financial Disclosures: None declared.
Acknowledgments: We acknowledge and appreciate the expert technical assistance of Frances Hayes, MD, Andrew Dwyer, RN, Joseph Moy, and Samir Aleryani, PhD, in the acquisition and handling of specimens at the MGH. L. M. Thienpont is indebted to Dietmar Stöckl for his help with the graphical and statistical interpretation of the method comparison data. S. Blincko acknowledges and appreciates the discussions with Jonathan Middle from the UK NEQAS, Birmingham, for suggestions towards the conduct of the study. M. M. Kushnir and A. L. Rockwood acknowledge ARUP Institute for Clinical and Experimental Pathology for support.
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Footnotes
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1 Nonstandard abbreviations: ID-LC, isotope dilution–liquid chromatography; MS/MS, tandem mass spectrometry; GC, gas chromatography; RMP, reference measurement procedure; JCTLM, Joint Committee for Traceability in Laboratory Medicine; LOQ, limit of quantification. 
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Abstract
Introduction
