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Clinical Chemistry 50: 2101-2110, 2004. First published September 16, 2004; 10.1373/clinchem.2004.037358
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(Clinical Chemistry. 2004;50:2101-2110.)
© 2004 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Evaluation of a Candidate Reference Measurement Procedure for Serum Free Testosterone Based on Ultrafiltration and Isotope Dilution–Gas Chromatography–Mass Spectrometry

Katleen Van Uytfanghe1, Dietmar Stöckl1, Jean M. Kaufman3, Tom Fiers3, H. Alec Ross4, André P. De Leenheer2 and Linda M. Thienpont1,a

1 Laboratory for Analytical Chemistry and 2 Laboratory for Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Gent, Belgium.
3 Laboratory for Hormonology and Department of Endocrinology, Ghent University Hospital, Gent, Belgium.
4 Department of Chemical Endocrinology, University Medical Centre, Nijmegen, The Netherlands.

aAddress correspondence to this author at: Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium. Fax 32-9-264-8198; e-mail linda.thienpont{at}ugent.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: To assess the analytical validity of free testosterone (FTe) measurements, a reference measurement procedure (RMP) is required. For steroids, isotope dilution–mass spectrometry is accepted as state-of-the-art technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the protein-bound fraction, the RMP should include a physical separation step. The use of equilibrium dialysis (ED) or ultrafiltration (UF) is advocated. Our objective was to develop such a candidate RMP.

Methods: We selected UF combined with isotope dilution–gas chromatography–mass spectrometry (ID-GC/MS) for direct measurement of Te in the ultrafiltrate. After optimization of the UF process, the complete procedure was validated by use of split-sample comparisons with indirect ED (iED) and symmetric dialysis (SyD).

Results: The candidate RMP gave maximum within-day, between-day, and total CVs of 3.0%, 3.1%, and 4.3%. The Deming regression equations for the respective method comparisons were: UF-ID-GC/MS = 0.98(iED) – 53 pmol/L (r = 0.94; Sy|x= 42 pmol/L) and UF-ID-GC/MS = 0.92(SyD) + 21 pmol/L (r = 0.97; Sy|x= 31 pmol/L).

Conclusions: We achieved the objective of a state-of-the-art candidate RMP, which agreed well with iED and SyD. However, we also demonstrated that a degree of discordance remains, which may require a decision from an authoritative organization on the recommended procedure to measure free hormone concentrations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
It is known that circulating testosterone (Te)1 exists in blood in the free (FTe) and protein-bound forms, with high affinity to sex-hormone-binding globulin (SHBG) and with low affinity to albumin (1). According to the free hormone hypothesis (2), FTe is more reflective of the physiologic actions of the hormone than the total testosterone (TTe) concentration. For this reason, the measurement of FTe concentrations has been promoted as the best diagnostic test to evaluate the androgen status of a patient. However, this supposes that the selected analytical method works under equilibrium conditions in vitro. The free/bound complex is a thermodynamic system, in which the FTe fraction depends on the total hormone and protein concentration as well as on protein association and dissociation rates, which in turn are influenced by temperature and pH (3)(4). Traditional measurement procedures separate FTe from the protein-bound Te fraction by equilibrium dialysis (ED) or ultrafiltration (UF). In some of these procedures, the FTe concentration is determined directly in the dialysate/ultrafiltrate, whereas others do it indirectly by first estimating the FTe fraction and relating it to the separately determined TTe concentration (5)(6)(7)(8)(9)(10). An alternative to ED and UF is symmetric dialysis (SyD). The interesting aspect of SyD is that it does not separate bound and free hormone because it works with serum at both sides of the membrane. The only process that takes place is redistribution of labeled hormone (11)(12). Thus, SyD is an indirect technique. Unfortunately, for routine use, ED, UF, and SyD procedures have been deemed to be technically too cumbersome. Therefore, single-step nonextraction FTe assays have been developed by the in vitro diagnostics industry. Alternatively, the FTe concentration is estimated by calculation based on the law of mass action from the concentration of TTe and relevant proteins or as the free androgen index (13)(14).

Considerable controversy has arisen about the analytical validity, in particular, of single-step nonextraction FTe assays (15)(16)(17). A major argument has been that the design of some assays insufficiently accounts for the law of mass action (3)(18). Although this problem has been documented in validation studies against a so-called gold standard, such as an analytical method with a valid physicochemical basis [see, for example, Refs. (14), (19)(20)(21)], these assays have continued to be marketed and used by the endocrine laboratory community. Recent European legislation, as decreed in the EC Directive on In Vitro Diagnostic Medical Devices (98/79/EC) (22) and supporting ISO/CEN standard (23), has provided a new strong incentive to bring analytical validation to the forefront. Indeed, since December 2003, a new commercial assay can obtain the CE label only after the diagnostic manufacturer has extensively documented the validation data. For well-defined analytes, this validation is to be done by split-sample measurements with a SI-traceable reference measurement procedure (RMP) [Note: in current metrologic terminology, the term gold standard is replaced by RMP (23)]. For steroid hormones such as Te, it is state of the art to use isotope dilution–mass spectrometry (ID-MS) as the RMP (24). This is because the theoretical measurement principle guarantees accurate, specific, and matrix-independent measurement and because ID-MS can be calibrated directly with gravimetrically weighed-in primary calibrator material. However, for FTe, ID-MS must be combined with a technique capable of separating free from bound hormone without equilibrium disturbance. In this respect, there is general agreement that ED and/or UF, at least in expert hands, are the best procedures (3).

From this perspective, the aim of our study was to develop such a candidate RMP. We opted for UF to separate free from bound Te and coupled it to ID–gas chromatography–MS (UF-ID-GC/MS) for direct measurement of Te in the ultrafiltrate. As part of the validation process, we performed the proposed measurement procedure in parallel with an indirect ED (iED) and SyD measurement procedure, as established in the laboratories of two of the authors, on a panel of 38 male sera.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
materials
Te standard material (with a purity >99.5% according to the manufacturer’s certificate) was purchased from Sigma-Aldrich. Isotope dilution was performed with [3,4-13C2]-Te from Cambridge Isotope Laboratories. Working solutions of both unlabeled and labeled Te (concentration, ~2.60 nmol/L) were diluted on a gravimetric basis from ethanolic stock solutions (~1040 µmol/L, pro analysis; Merck). The quality of the other solvents was gradient grade (methanol; Romil), ultrapure (water; 18.2 {Omega}; produced with an Elga Elgastat Maxima Analytical water purification system), pro analysis (formic acid; Fluka), or dried (cyclohexane; Merck). The HEPES buffer (52.75 mmol/L), pH 7.4, contained 5.265 g/L NaCl, 0.224 g/L KH2PO4, 0.275 g/L MgSO4 · 7 H2O, 12.570 g/L HEPES, 0.3 g/L urea, 0.275 g/L CaCl2 · 2 H2O, 0.9 g/L NaOH, and 0.520 g/L NaN3 (all from Sigma-Aldrich). For UF, Millipore Centricon YM-30 and YM-10 UF devices (cutoffs of 30 and 10 kDa, respectively; membrane of regenerated cellulose) were used. In addition, three different UF device types from Vivascience were investigated: the Vivaspin 2 variant with membranes of regenerated cellulose, polyethylenesulfone, or cellulose acetate, all with a 10-kDa cutoff. The membranes in the Centricon devices were horizontally placed, whereas those in the Vivaspin devices were vertically mounted. Solid-phase extraction was done with Waters Oasis HLB 1-mL (30 mg) extraction cartridges. The heptafluorobutyric anhydride derivatization reagent was from Macherey and Nagel. For internal accuracy control, a lyophilized serum sample [with a TTe target value of 19.40 nmol/L, as determined by an ID-GC/MS RMP (25)] was purchased from the German Society of Clinical Chemistry and Laboratory Medicine.

instrumentation
Serum was ultrafiltered in a thermostatically controlled, fixed-angle centrifuge (Sorvall RT7 plus with a SL-50RT rotor). The solid-phase extraction cartridges were mounted on a JT Baker SPE-12G Column Processor® device. For HPLC, a SpectraSystem P1000 pump (ThermoSeparation Products) with a variable-wavelength ultraviolet detector (Philips) was used. The HPLC was equipped with a Hypersil BDS C18 column [150 x 2.1 mm (i.d.); 5 µm bead size; Alltech]. GC-MS was performed with a Finnigan MAT Incos XL mass spectrometer combined with a 5890 Series II GC (Hewlett Packard). The GC fused-silica column was a DB-1, i.e., methylsilicone type [20 m x 0.18 mm (i.d.); 0.4 µm film thickness; J&W Scientific]. The GC was equipped with a programmable temperature vaporizing injection system (Gerstel GmbH). Densities were measured with a Mettler Toledo DA-110M density meter.

measurement procedures
UF-ID-GC/MS.
To 1 mL of each serum sample, directly pipetted into a Centricon YM-30 UF device, 1.4 mL of HEPES buffer was added. All volumetric steps that would contribute to method accuracy were gravimetrically controlled. During equilibration of the mixture at 37 °C for a minimum of 30 min, the centrifuge was preheated by rotating at 3355g for 1 h until a temperature of 37 ± 1 °C was reached. The samples were then immediately centrifuged at 2402g for 16 min, which yielded, on average, 800 µL of ultrafiltrate. To the ultrafiltrate, [3,4-13C2]-Te was added in an amount equivalent to the ultrafiltered endogenous Te to obtain a 1:1 isotope ratio on MS determination. This required that before starting the analysis, the FTe concentrations in the samples had been roughly determined. The mixture was again allowed to equilibrate for 1 h. Meanwhile, the Oasis HLB cartridges were conditioned by wetting consecutively with 1 mL of methanol and water. Immediately after the conditioning step, the ultrafiltrate, acidified with 40 µL of 50 mL/L formic acid, was applied and passed through the cartridge at a flow rate of ~0.4 mL/min. The cartridge was washed with, respectively, 1 mL of water and 1 mL of a methanol–water mixture (60:40 by volume). Te and its [3,4-13C2]-analog were subsequently eluted with 1 mL of a methanol–water mixture (90:10 by volume). The eluate was evaporated to dryness at 50 °C under N2. The residue was redissolved in 50 µL of the HPLC mobile phase and injected on the HPLC column. Isocratic chromatography was performed with a mixture of methanol–water (60:40 by volume) at a flow rate of 0.2 mL/min. The fraction containing Te and [3,4-13C2]-Te (eluting between 8.50 and 11.50 min) was collected and evaporated at 50 °C under N2. The evaporated residue was redissolved in 20 µL of cyclohexane, and 20 µL of heptafluorobutyric anhydride was then added and allowed to react for 30 min at 70 °C. After evaporation of the reaction mixture at room temperature under N2, ~12 µL of cyclohexane was added, from which 6 µL (0.12 pmol) was injected in the solvent vent mode (70 °C). After 0.3 min, the split was closed, and the temperature of the injector increased to 280 °C at a rate of 12 °C/s. After 2 min at 280 °C, the injector was cooled to the initial temperature. The helium column head pressure was 12 psi. The GC oven was programmed for the following temperature gradient: hold at 150 °C for 1.3 min, then increase to 300 °C (ramp rate, 20 °C/min) and hold for 3 min. The column was then heated at 325 °C for 8.5 min. Both the derivatized Te and [3,4-13C2]-Te eluted at 10.45 min. For MS detection in the selected-ion monitoring mode (SIM), the molecular ions of the diheptafluorobutyryl derivatives of both unlabeled and labeled Te were monitored at m/z 680 and 682, respectively. Dwell times were chosen to obtain at least 15 cycles under the peaks.

For calibration, equivalent amounts of Te and [3,4-13C2]-Te (~0.35 pmol of each) were sampled on a gravimetric basis to obtain a 1:1 isotope ratio. The mixtures were evaporated to dryness and derivatized as described for the processed serum samples. The ID-MS one-point calibration protocol is described in detail elsewhere (26).

iED.
iED was performed in Membra-CelTM MD 10-14 tubing as described previously (14)(27). Briefly, 125 µL of plasma containing a trace amount of [1,2,6,7-3H]-Te, diluted 1:5 with saline, was dialyzed under continuous shaking for 15 h at 37 °C against 5 mL of saline. After equilibrium was reached, 0.5 mL each of diluted plasma and the dialysate was added to separate vials containing 10 mL of liquid scintillation mixture and counted. The free fraction was calculated according to Slaunwhite and Sandberg (28). The purity of the tracer was checked every 4 months, and if necessary, the tracer was purified by paper chromatography.

SyD.
SyD was performed in a Dianorm dialysis apparatus with Visking dialysis membrane, as described elsewhere (11). Briefly, 500 µL of each sample was incubated for 30 min at 37 °C with a trace amount of paper-chromatography-purified [1,2,6,7-3H]-Te. After incubation, 180 µL of the incubate was pipetted into one compartment of a temperature-equilibrated dialysis cell, and the other compartment was filled with an equal volume of the same serum sample without tracer. Dialysis was performed for 2.5 h at 37 °C. Subsequently, 100 µL of the samples in each compartment was counted by liquid scintillation, and the free fraction was calculated as described previously. Calibration of the dialysis system was performed by assessment of membrane permeability with use of a calibrator serum (normal plasma with 100 nmol/L Te added) in which the FTe fraction had been established by direct ED-ID-GC/MS.

TTE measurement
Measurement of the TTe concentration was performed according to our previously published candidate RMP (29). In short, to a certain volume of serum, representing 2.6 nmol of Te, was added an equivalent amount of [3,4-13C2]-Te, and the mixture was allowed to equilibrate. The sample was then extracted with dichloromethane. After centrifugation, the organic phase was transferred to another vial for evaporation to dryness under N2. The residue was fractionated sequentially by SephadexTM LH-20 chromatography and HPLC. The final collected fraction was evaporated to dryness under N2, derivatized, and analyzed by GC-MS as described for FTe.

The measurement procedure was performed according to preset analytical specifications: a maximum total CV (CVT) of 2.0% and maximum systematic deviation from the target of a certified reference material of 0.9% (30).

method-comparison study
UF-ID-GC/MS was compared with iED and SyD by split-sample measurement of 38 serum samples. The UF-ID-GC/MS measurement protocol consisted of analyzing each sample in two independent runs, which means that in each run, separate calibration and sample pretreatment were used. In total, nine measurement series were needed to process all samples twice. For iED and SyD, duplicate measurements were performed, but in the same run. This protocol gave a total of two measurement series for iED and six for SyD. For calculation of the FTe concentration from the fractions determined by iED and SyD, the TTe concentrations as obtained by the ID-GC/MS candidate RMP were used.

serum samples
The sera used for the method-comparison study were obtained from single blood donations from 38 apparently healthy males. Sera were purchased from DiaServe Laboratories GmbH. Serum was isolated by centrifugation 1 h after collection. The sera were stored at 4 °C for 3 days, filtered, and fractionated into 1-mL portions in Eppendorf vials. The aliquots were frozen and sent on dry ice to the laboratory in Ghent. The sera were also kept on dry ice during transport from Ghent to the other participating laboratories. Receipt of the samples in the frozen form was confirmed. Until analysis, in each laboratory specimens were stored at –20 °C. All samples were characterized for SHBG (with the SHBG immunoradiometric assay from Orion Diagnostica) and albumin (with N antiserum to human albumin; Dade Behring Inc.).

imprecision and internal accuracy control during the method comparison
To control the between-run reproducibility of the UF procedure during the split-sample measurements, internal quality control (IQC) was performed. It consisted of repeated duplicate analysis of two serum samples with FTe concentrations of 138.7 and 431.7 pmol/L, respectively. Because the number of samples analyzed per analytical run surpassed the capacity of the centrifuge (two centrifugal runs were needed for the UF process), we split the duplicates of each serum between the two centrifugal runs. This IQC protocol was performed in a total of 20 runs, including the 9 series needed for completing the method comparison to calculate the CVT and the between- and within-day CVs (CVdd and CVwr, respectively) according to the NCCLS EP-5 protocol (31). For internal control of the ID-GC/MS measurements after UF, a lyophilized IQC sample gravimetrically diluted with 9 g/L NaCl to a concentration of 350.7 pmol/L, was analyzed in duplicate in each run. These results were evaluated in terms of the mean deviation from the target value and the CVT. For the measurements using iED and SyD, the standard operating procedures of the respective laboratories were followed, which included in-house IQC samples.

statistics and graphics used in the method-comparison study
The results of the method-comparison study are represented graphically as scatter plots (iED or SyD on the x axis, UF-ID-GC/MS candidate RMP on the y axis). Deming regression analysis was performed for the different method pairs, using CBstat software (Ver. 4.3.2; from K. Linnet, Risskov, Denmark). The 95% prediction interval was calculated as 1.96 times the CVs of the method pairs (CVmp). The latter were calculated with the following formula:

In this equation, CVA represents the within-run imprecision of either the iED or SyD procedure and was calculated from the duplicate measurements of the serum samples according to the formula:

, where d represents the difference between the duplicates, n is the number of duplicates, and is the mean of the serum concentrations. The CVB in the equation for CVmp represents the imprecision for the UF-ID-GC/MS candidate RMP and was calculated using the difference between the duplicates performed in two analytical runs.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
uf-id-gc/ms candidate rmp
For the development of our UF-ID-GC/MS candidate RMP, we started from the TTe equivalent (29). Because GC/MS measurement of Te can be performed with a limit of detection of 2 pmol/L (signal-to-noise ratio of 3), we considered this procedure appropriate for quantification of FTe in sera from apparently healthy males (FTe concentration range observed in this study, 106–640 pmol/L). However, to achieve specifications appropriate for a RMP, the signal-to-noise ratio at which quantification is performed is important. We set the limit at ~25. Whereas for serum FTe concentrations of 100 pmol/L and processing of 0.8 mL of ultrafiltrate, we achieved a ratio of ~50–70, concentrations of 15–20 pmol/L necessitated processing of 1.6 mL of ultrafiltrate to reach our limit, but with increasing imprecision. Because working with a higher volume of ultrafiltrate was not considered practicable, we estimated the limit of quantification of our current procedure at 15–20 pmol/L. The only variant we introduced was substitution of the liquid–liquid extraction and Sephadex chromatography by solid-phase extraction with Oasis HLB cartridges. The reconstructed ion chromatograms after solid-phase extraction and HPLC were as clean and interference-free as obtained with our traditional procedure (29). Nevertheless, we investigated possible interference by other steroid hormones (Table 1 ). As indicated in Table 1 , interference by some steroids could immediately be excluded on theoretical grounds because the relative molecular weights of the possible heptafluorobutyryl derivatives were lower than the m/z values monitored for Te and [13C2]-Te. The other steroid hormones were derivatized under exactly the same conditions as optimized for Te and subjected to GC/MS analysis in both the scan (20 ng of derivatized substance injected) and SIM mode (2 ng, except for epitestosterone, for which only 150 pg was injected). As expected, epitestosterone derivatized in exactly the same way. However, the derivative of the Te isomer did not interfere because its relative retention time was 0.93. For (3ß,17ß)-5{alpha}-androstan-3,17-diol and (3ß,17ß)-androst-5-ene-3,17-diol, we observed from the registered spectra that they also yielded a diheptafluorobutyryl derivative with molecular ions at m/z 684 and 682, respectively. The derivatives eluted in our GC program with relative retention times of 1.02 and 1.00, respectively. Although on this basis the derivative of (3ß,17ß)-5{alpha}-androstan-3,17-diol did not interfere, we confirmed this fact by performing SIM at m/z 682 and 680. With respect to the derivative of (3ß,17ß)-androst-5-ene-3,17-diol, we observed that, because of the suboptimal derivatization conditions (they were optimized for Te), the signal abundance in SIM for 2 ng was very low in comparison with that for Te. For that reason, we injected decreasing amounts of the derivative and found that at <500 pg the signal-to-noise ratio was already <3. On this basis, we assume that interference after processing of a serum sample containing physiologic concentrations of (3ß,17ß)-androst-5-ene-3,17-diol would be very unlikely (32).


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  		Table 1. Steroid hormones tested for possible interference on SIM at m/z 680 and 682 (molecular ions of the diheptafluorobutyryl derivatives of Te and [13C2]-Te).

IQC of the ID-GC/MS measurement procedure during 20 measurement series showed a mean deviation from the target of –0.5% and an associated CVT of 1.1%, a CVdd of 0.4%, and a CVwr of 1.0% (Table 2 ). From these data we concluded that our slightly modified RMP demonstrated adequate trueness and precision and that no further validation of the ID-GC/MS measurement procedure was necessary.


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  		Table 2. Precision data for the UF-ID-GC/MS candidate RMP in comparison with iED and SyD.

For separation of TTe from protein-bound Te, we selected UF on the basis of the speed and ease of performance. Devices with five different membrane types and/or size cutoffs were evaluated (Table 3 ). Selected criteria were the degree of protein leakage, determined with the Coomassie® Plus Protein assay from Pierce in the ultrafiltrate from 1 mL of serum (n = 8), and adsorption of Te, estimated from recovery experiments after UF of a 9 g/L NaCl solution to which 433 pmol/L Te had been added (n = 5) (33). We defined the limit for protein leakage on the basis of the error the leakage would induce in the FTe measurements, with 1% error considered allowable. Assuming a total protein concentration of 70 g/L and a mean FTe concentration of 2.6% (34), we allowed a maximum of 0.025% protein leakage. As can be seen from Table 3 , all tested ultrafiltration devices fulfilled this protein leakage criterion.


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  		Table 3. Characteristics of the tested UF devices in terms of protein leakage and adsorption.

With respect to the adsorption studies, the results showed best performance by the Centricon YM-30 device, which was in perfect agreement with the recommendations on UF for free thyroid hormones (33)(35). With respect to the 2% adsorption of Te observed from the saline solution with this device (Table 3Up ), we tested whether the adsorption took place by contact with the serum or with the ultrafiltrate. In the first case, this would not influence the FTe measurement results because of the thermodynamic equilibrium between TTe and FTe. These investigations were done by adding tritiated Te to undiluted and 1:1.5-diluted serum. (Although small dilutions reduce the TTe concentration, they have little effect on the FTe concentration.) This experiment showed a fourfold higher adsorption from undiluted serum, whereas less ultrafiltrate was generated (550 µL) compared with the diluted sample (700 µL). From this we concluded that adsorption of Te indeed was attributable to contact with the serum. Although it was not possible to monitor each UF device for protein leakage or adsorption, we verified by use of random controls with repeated analysis of two human sera in duplicate whether there was sufficient evidence for the consistency of the UF process in terms of absence of protein leakage and adsorption (see discussion of IQC results below). These IQC measurements also allowed us to study the lot-to-lot variation of the UF devices, as has been described for free thyroxine (33).

To avoid disturbing the equilibrium between bound and free Te, we paid attention to the temperature and pH, which are known to influence UF (4). However, the risk of equilibrium disturbance also depends on the duration of the UF process, which in turn depends on the viscosity of the serum and thus on the dilution and the UF yield. The Donnan effect (36)(37), which also depends on the initial sample volume and pH, should be kept as low as possible. From this perspective, we performed several experiments to optimize the UF process. With respect to temperature, UF should be done at 37 °C; however, even with a thermostatically controlled centrifuge, it is necessary to ensure that this temperature is reached from the beginning of the centrifugal process onward because it is in the initial phase that the yield of ultrafiltrate is the greatest. We observed that we had to preheat the empty centrifuge by rotating the rotor at 3355g for ~1 h to obtain a temperature of 37 ± 1 °C. Under these conditions, we confirmed the temperature-related difference (4)(8)(9); there was an increase in FTe concentration of ~2.8% for every 1°C that the temperature was closer to 37 °C (P <1.32 x 10–10). Because of the effect of pH, it was necessary to ensure that before UF, the serum was at a physiologic pH of 7.4. We used HEPES buffer for this purpose, either by adding 50 µL of a concentrated buffer solution (185 g/L HEPES, 8.8 g/L NaOH) to 1.0 mL of serum before UF or by diluting the serum with the 52.64 mmol/L HEPES buffer as described earlier. The latter way of adjusting the pH was considered best because of our experience indicating that diluted serum needs shorter UF times with a higher UF yield. To test the maximum allowable dilution without alteration of the FTe concentration (9)(38)(39), we compared three different dilutions with undiluted serum. As can be seen from Table 4 (values under "dilution of serum with buffer"), there was a significant difference, amounting to 13.7%, only after addition of 9 mL of buffer (dilution 1:10). This dilution effect agrees with predictions that can be made on the basis of the law of mass action. We therefore decided to perform all further experiments and the final UF procedure using the 1:2.4 dilution with the 52.75 mmol/L HEPES buffer. With respect to the Donnan effect, we investigated the relationship between the volume of sample subjected to the UF process and the yield of ultrafiltrate. As can be seen from Table 4 , on processing of 1.0, 1.7, and 2.4 mL of diluted (1:2.4) serum and generating an ultrafiltrate yield of ~33%, we found no significant difference in FTe concentration. The data in Table 4 indicate that a yield of ~17% is too low, whereas a yield of ~33% is sufficient. On the basis of these experiments, we opted to use a sample volume of 2.4 mL (1 mL of serum + 1.4 mL of buffer) for UF, which would generate ~0.8 mL of ultrafiltrate and would give results above the limit of detection of our ID-GC/MS procedure.


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  		Table 4. Optimization of the UF process in terms of dilution of the serum, sample volume on the membrane, and generated volume of ultrafiltrate.

With respect to the precision of the UF-ID-GC/MS candidate RMP, the IQC results according to the NCCLS EP5-A protocol (Table 2Up ) demonstrate an acceptable precision in terms of CVwr, CVdd, and CVT. These CVs also include the variability of the UF process and, thus, give sufficient evidence for its reproducibility and the absence of lot-to-lot variation in the UF devices (33). For the four different lots used to date, we have not observed any unacceptable variation. However, because the latter is a risk (33), we recommend validating the performance of each new lot before use with at least two IQC samples with known target concentrations. For comparison, Table 2Up shows the CVT values for iED and SyD. The data are slightly in favor of UF-ID-GC/MS. The same applies for the CV values calculated from the duplicate results of the serum samples, i.e., 3.3% vs 5.6% and 7.6% for iED and SyD, respectively. The mean CVT as calculated for UF-ID-GC/MS (3.7%) was used to calculate the acceptance criterion during the method-comparison study, namely, to predict the maximum difference between duplicates determined on two separate occasions, i.e., (1.96 x xCVT), or 10.3%. The largest difference we observed was 6.8%; therefore, no UF-ID-GC/MS measurements for any of the 38 serum samples had to be repeated.

method-comparison study
To validate the newly developed UF-ID-GC/MS candidate RMP, we performed split-sample measurement of 38 sera against iED and SyD. Because iED and SyD are indirect and measure the FTe fractions (%), the corresponding concentration was calculated by multiplication of the TTe concentration. The laboratories in this study determine TTe concentrations with a RIA. However, in view of the fact that in this study the procedures for estimating the FTe fraction were of interest, rather than differences in TTe assays, as discussed recently in the literature (40)(41), we used the TTe concentrations as determined with our SI-traceable TTe candidate RMP (29). These concentrations ranged from 7.6 to 47.2 nmol/L, with a mean of 16.7 nmol/L.

Scatter plots of the FTe results obtained by UF-ID-GC/MS vs those obtained by iED and SyD are shown in Fig. 1 . The plots also include the line of equality, the Deming regression line, and the 95% prediction interval. The corresponding outcomes of the Deming regression analysis are summarized in Table 5 . The serum FTe concentrations measured by UF-ID-GC/MS ranged from 106 to 640 pmol/L, with a mean of 299.4 pmol/L, and corresponding FTe fractions of 1.1–2.8% (mean, 1.8%) of the TTe. For iED and SyD, the FTe fractions ranged from 1.3% to 3.3% (mean, 2.2%) and from 1.2% to 4.4% (mean, 1.8%), respectively, giving FTe concentrations of 128–660 pmol/L (mean, 360.4 pmol/L) and 122–750 pmol/L (mean, 301.9 pmol/L), respectively.



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  		Figure 1. Measurements results (pmol/L) for serum FTe obtained by UF-ID-GC/MS compared with those by iED (A) and SyD (B).

The solid line indicates the Deming regression; the dotted lines indicate the 95% prediction interval, and the dashed line indicates the line of identity (x = y).


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  		Table 5. Summary of the Deming regression analysis data for the method-comparison study.

As shown in Table 5Up , UF-ID-GC/MS correlates fairly well with both iED and SyD. However, there is a proportional difference between UF-ID-GC/MS and iED or SyD, with a negative intercept for iED and a positive intercept for SyD, both significantly different from zero. From these data, one can infer that iED and SyD also correlate well, but with a constant systematic difference. A comparison of the Sy|x values shows that they are highest for the comparison of UF-ID-GC/MS with iED. Although most of the spread around the regression line can be explained by the CVT (most data are within the 95% prediction limits), these observations are presumptive that iED might be most susceptible to individual sample matrix-related effects. Further visual inspection of all scatter plots shows that in Fig. 1BUp , the 95% prediction interval best includes the line of equality with almost symmetric location of the data pairs along that line. This indicates that our UF-ID-GC/MS candidate RMP agrees best with SyD. However, there are more data pairs outside the prediction limits than would be statistically expected, which could be the result of some degree of sample-related effects in one of the measurement procedures.

The method comparisons show that in the absence of an absolute gold standard, it is not possible to conclude which of the three procedures gives the "true" FTe concentration. Therefore, it might be appropriate here to consider the degree of independence of the three procedures, which is indeed a hallmark of a SI-traceable RMP. With respect to our candidate UF-ID-GC/MS RMP, we showed that direct measurement of the FTe concentrations in ultrafiltrates is feasible. This contrasts with iED and SyD, which cannot be claimed as RMPs because of the indirect measurement principle. The problem with UF, however, is the uncertainty as to whether the measured concentration is the same as the FTe concentration in the original sample. Technical problems such as protein leakage, unequal distribution of FTe resulting from the Donnan effect, and failure to keep the temperature at 37 °C can be ruled out for UF, as shown from the IQC data. A potential problem lies in the fact that during UF, unfilterable constituents of the serum become concentrated, including those that may possess positive or negative cooperative effects toward the interaction between Te and its binding proteins. The finding that moderately diluting serum with buffer has virtually no effect on the measured FTe demonstrates that, if present, such effects are probably quantitatively unimportant. The nature of the dilution buffer might be crucial in this respect. If we consider the measurement principle of iED, where the tracer that is present at equilibrium in the dialysate is determined relative to the total amount added to the sample, the main effect on accuracy is tracer impurity. However, this can be minimized by dilution, which at the same time will reduce the Donnan effect to negligible proportions. Dilution must be taken into account when estimating the FTe fraction for undiluted serum because this quantity must be multiplied by an independently measured TTe concentration to obtain an estimate of the FTe concentration. Theoretically, the particular proportions of Te, SHBG, and albumin and their affinities in serum allow for an accurate result when optimal algorithms are used to correct even for high dilutions. Nevertheless, the same restrictions as those mentioned above for UF with regard to the absence of undialyzable components affecting Te-protein interaction and buffer composition apply. Lastly, SyD is not affected by undialyzed substances and the Donnan effect discussed above for UF and ED because the same sample is present on both sides of the membrane and no physical separation of free and bound Te takes place. Therefore, the FTe concentration in the original sample is not altered in SyD. The effect of tracer impurities is also so small that dilution is not necessary. However, to translate the dialysis rate into the free fraction, SyD needs to be calibrated by either direct UF or ED. This is probably the biggest hurdle to the trueness of SyD measurements because an error in the assignment of the free fraction for the calibrator serum propagates as a systematic error in all results. Therefore, to evaluate which of the three procedures most closely approximates the ideal, one has to balance the uncertainties inherent to each of them. In UF-ID-GC/MS, the only uncertainty remaining is independence of dilution as an indicator of the absence of effects of unfilterable substances. Although demonstrated for the present candidate RMP on 38 sera from apparently healthy males, this needs extension to different kind of samples. The uncertain factor in iED is the extent to which the necessary correction for dilution follows the theoretical model for a larger variety of samples. For SyD, although the above uncertainties do not exist, the uncertainty lies in the calibration. However, in this study, the validity of the calibration process was ensured assigning the value by use of a generally accepted RMP (ID-GC/MS) (24).

In summary, the present study shows that we were able to develop a state-of-the-art candidate RMP based on UF in combination with ID-GC/MS for direct measurement of FTe in sera from males. When we challenged the UF-ID-GC/MS candidate RMP in a method-comparison study with 38 male sera against two indirect "gold standard" methods, UF-ID-GC/MS showed good correlation and acceptable agreement between results. Nevertheless, small systematic differences and sample-related effects were observed. From these observations and theoretical considerations about the independence of the measurement procedures used, it is clear that even the best available and scrupulously performed measurement procedures have technical and fundamental limitations and that, consequently, the scientific community will have to accept that there will remain a degree of arbitrariness about the best way to measure free hormone concentrations. It will be up to authoritative organizations such as the IFCC (43) and the Joint Committee on Traceability in Laboratory Medicine (44) to decide on which measurement procedure should be used by the in vitro diagnostics industry in establishing metrologic traceability of routine assays for serum free hormones. Last but not least, the next challenge for our laboratory will be optimization of our candidate RMP to achieve sufficient sensitivity for measurement of FTe concentrations in females with androgen deficiencies.


   Acknowledgments
 
We gratefully acknowledge financial support by the Fund for Scientific Research-Flanders G.0038.01.


   Footnotes
 
1 Nonstandard abbreviations: FTe and TTe, free and total testosterone, respectively; SHBG, sex-hormone-binding globulin; (i)ED, (indirect) equilibrium dialysis; UF, ultrafiltration; SyD, symmetric dialysis; RMP, reference measurement procedure; ID-GC/MS, isotope dilution–gas chromatography–mass spectrometry; SIM, selected-ion monitoring; and IQC, internal quality control.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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