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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
LabCorp Vs Quest free testosterone lab ranges: Why are they different?
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<blockquote data-quote="madman" data-source="post: 216090" data-attributes="member: 13851"><p><strong>Direct measurement of serum-free testosterone by ultrafiltration followed by liquid chromatography-tandem mass spectrometry (2009)</strong></p><p><strong></strong></p><p><strong></strong></p><p><strong>Background: </strong><em>Currently there is no reliable method suitable for routine measurement of serum-free testosterone (FT).</em></p><p></p><p><strong>Aim:</strong> <em>To develop such a method involving liquid chromatography-tandem mass spectrometry (LC-IDMS/ MS) that directly detects and quantifies the FT present in serum.</em></p><p></p><p><strong>Methods:</strong> <em>Ultrafiltrate testosterone obtained from 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analog FT immunoassay, free testosterone calculated from mass action equations (cFT), and with equilibrium dialysis (ED) coupled LC-MS/MS.</em></p><p></p><p><strong>Results:</strong> <em>Total imprecision determined over twenty runs was b6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analog immunoassay (correlation coefficient 0.667; bias −81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LCMS/MS (correlation coefficient 0.8996, bias 10%). <u>The agreement was closest with the ED method (correlation coefficient 0.9779, bias 2.4%)</u>.</em></p><p></p><p><strong>Conclusion:</strong> <em>We present a relatively simple UF coupled LC-MS/MS definitive method that measures serum-free testosterone. The method is relatively fast, reliable, and is suitable for routine clinical laboratory practice.</em></p><p></p><p></p><p></p><p></p><p><strong><em>Several approaches have been used to measure FT in circulation. The most reliable method physically separates the protein-bound from the free testosterone prior to quantifying the latter either through indirect measurement involving radioactively labeled tracer or direct measurement. The physical separation has traditionally been carried out by equilibrium dialysis (ED), a tedious technique for routine clinical practice [3]. Also problematic, tracer impurities can cause substantial errors when a radioactively labeled tracer is used to indirectly quantify the free fraction.</em></strong><em> A second approach has sought to calculate the free fraction from the amount of total testosterone, the binding capacity of SHBG and albumin, and the affinity constants of albumin and SHBG for testosterone. The calculated FT (cFT) usually correlates well with FT measured by the reference equilibrium dialysis method but is highly dependent on the accuracy of the total testosterone, SHBG, and albumin quantification [3,13,14].<strong> The final approach, most widely used in clinical labs but fraught with inaccuracy, has utilized analog-based immunoassay to estimate the free fraction. Unfortunately, estimates by this approach reflect total testosterone levels more closely than they do the free fraction [6,15].</strong></em></p><p><em><strong></strong></em></p><p><em><strong>Recently, Van Uytfanghe et al. [16,17] reported a reference method for FT that separated the protein-bound and free fractions by ultrafiltration (UF) instead of by equilibrium dialysis. This was attractive insofar as <u>ultrafiltration is inherently faster and less technically demanding than equilibrium dialysis</u>. However, the solid phase purification and the GC-MS detection used by Van Uytfanghe et al. is cumbersome and time-consuming which makes it difficult for routine clinical testing.</strong> Previously we reported an LC-MS/MS procedure [4] for the measurement of serum total testosterone.<strong> Here we describe a <u>new method using UF coupled with our testosterone LCMS/MS procedure for the measurement of FT offering further improvements in analytical sensitivity, convenience, and decreased sample requirement</u>. A split sample comparison against analog immunoassay, cFT, and ED coupled LC-MS/MS is also presented.</strong></em></p><p></p><p></p><p></p><p></p><p><strong>Discussion</strong><em> </em></p><p><em></em></p><p><em>Reliable measurement of free steroid and thyroid hormones in the blood is inherently technically challenging and until recently has been difficult to achieve in routine clinical practice. <strong>However, the increased sensitivity and ease of use of LC-MS/MS technology has made this an attractive, if not superior, alternative to immunoassay, and when coupled to prior ultrafiltration (UF) or equilibrium dialysis (ED) to remove the protein-bound fraction, a powerful tool to measure free hormone and drug levels.</strong> Several methods using just this technology have recently been published for the measure of free thyroid hormone and unbound antiretroviral drugs [23-25]. <strong>Although both UF and ED are acceptable as reference procedures to separate protein-bound from circulating free ligands, <u>UF is inherently better suited to the demands of the clinical lab because of its greater simplicity and speed, and accordingly, we chose to implement it in our proposed method</u>. <u>To the best of our knowledge, our proposed method is the first in the literature that directly measures FT by LC-MS/MS</u>.</strong></em></p><p><em><strong></strong></em></p><p><em><strong>*The UF-LC-MS/MS method described here compares favorably to the UF coupled GC-MS reference method recently published by Van Uytfanghe et al. [16]. This method achieves equivalent LLOQ (16 pmol/L vs. 15–20 pmol/L) with less sample (0.5 mL vs. 1 mL serum), higher throughput (3.9 vs. 10.45 min LC elution), and no need for derivitization.</strong></em></p><p><em><strong></strong></em></p><p><em><strong>The Centrifree® ultrafiltration device is designed for the rapid separation of free ligands by the unhindered passage through the YM hydrophilic and nonabsorptive membrane (30 kDa cutoff) with a high degree of protein retention. <u>This membrane was ideal in that it truly retained the protein present in the serum (less than 0.07 g/L, the limit of detection of our protein assay, in the ultrafiltrate) while not adsorbing FT (Table 1, Supplementary Data)</u>. Of note, the Centrifree® ultrafiltration device only worked when spun in a fixed angle rotor; swinging bucket rotors were unsuitable.</strong></em></p><p><em><strong></strong></em></p><p><em><strong></strong></em></p><p><em><strong></strong></em></p><p><em><strong></strong></em></p><p><em><strong><u>Our proposed method is attractive in that it agrees closely with ED and LC-MS/MS which must be considered the gold standard, but is much faster and easier to run, and better suited for the routine clinical laboratory, than the latter</u>. <u>The ultrafiltration procedure requires approximately 1 h to complete while equilibrium dialysis requires at least 16 h to complete the dialysis step</u>. The dynamic range of the procedure described here easily accommodates FT levels expected in the adult male, and lipemia, icterus, and other steroids do not interfere, and imprecision is less than 6% at FT levels expected in the adult male.</strong> The limitation of the current LLOQ makes the method is not sufficient for all female samples at low levels. However, for female test application, it is the high end of the range most clinical interested, i.e. hyperandrogenic conditions such as polycystic ovary syndrome. The 16 pmol/L LLOQ needs to be reduced by at least half to make the method suitable to measure with the imprecision of less than 20% of the FT levels expected in many women. Reducing the functional sensitivity by 50% might be achieved by increasing the serum ultrafiltrate from 0.5 to 1 mL or by adopting atmospheric pressure photoionization, but these will require additional studies to substantiate.<strong> T<u>he overall fraction of total testosterone concentration measured by the present UF-LC/MS/MS method is 1.72%, which is very close to the reported before as 1.87% by UFGC/MS</u> [17]. Further clinical studies are needed for the evaluation of clinical utilities of FT by this UF coupled LC-MS/MS method in subpopulations such as hypoandrogenic men and polycystic ovary syndrome women.</strong></em></p><p></p><p></p><p></p><p></p><p><strong>*<em>In conclusion, we have developed a simple, rapid, highly selective, and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput, and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice.</em></strong></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]19270[/ATTACH]</p><p>[ATTACH=full]19271[/ATTACH]</p><p><strong>Fig. 4. Method comparison of UF coupled LC-MS/MS with ED coupled LC-MS/MS (n= 26). The upper panel shows the correlation plot of UF vs. ED. The lower panel shows plots of the bias between free testosterone measurements by UF and ED as a percentage of ED values (Y-axis) against the ED measured FT concentration. UF, ultrafiltration coupled LC-MS/MS; ED, equilibrium dialysis coupled LC-MS/MS.</strong></p></blockquote><p></p>
[QUOTE="madman, post: 216090, member: 13851"] [B]Direct measurement of serum-free testosterone by ultrafiltration followed by liquid chromatography-tandem mass spectrometry (2009) Background: [/B][I]Currently there is no reliable method suitable for routine measurement of serum-free testosterone (FT).[/I] [B]Aim:[/B] [I]To develop such a method involving liquid chromatography-tandem mass spectrometry (LC-IDMS/ MS) that directly detects and quantifies the FT present in serum.[/I] [B]Methods:[/B] [I]Ultrafiltrate testosterone obtained from 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analog FT immunoassay, free testosterone calculated from mass action equations (cFT), and with equilibrium dialysis (ED) coupled LC-MS/MS.[/I] [B]Results:[/B] [I]Total imprecision determined over twenty runs was b6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analog immunoassay (correlation coefficient 0.667; bias −81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LCMS/MS (correlation coefficient 0.8996, bias 10%). [U]The agreement was closest with the ED method (correlation coefficient 0.9779, bias 2.4%)[/U].[/I] [B]Conclusion:[/B] [I]We present a relatively simple UF coupled LC-MS/MS definitive method that measures serum-free testosterone. The method is relatively fast, reliable, and is suitable for routine clinical laboratory practice.[/I] [B][I]Several approaches have been used to measure FT in circulation. The most reliable method physically separates the protein-bound from the free testosterone prior to quantifying the latter either through indirect measurement involving radioactively labeled tracer or direct measurement. The physical separation has traditionally been carried out by equilibrium dialysis (ED), a tedious technique for routine clinical practice [3]. Also problematic, tracer impurities can cause substantial errors when a radioactively labeled tracer is used to indirectly quantify the free fraction.[/I][/B][I] A second approach has sought to calculate the free fraction from the amount of total testosterone, the binding capacity of SHBG and albumin, and the affinity constants of albumin and SHBG for testosterone. The calculated FT (cFT) usually correlates well with FT measured by the reference equilibrium dialysis method but is highly dependent on the accuracy of the total testosterone, SHBG, and albumin quantification [3,13,14].[B] The final approach, most widely used in clinical labs but fraught with inaccuracy, has utilized analog-based immunoassay to estimate the free fraction. Unfortunately, estimates by this approach reflect total testosterone levels more closely than they do the free fraction [6,15]. Recently, Van Uytfanghe et al. [16,17] reported a reference method for FT that separated the protein-bound and free fractions by ultrafiltration (UF) instead of by equilibrium dialysis. This was attractive insofar as [U]ultrafiltration is inherently faster and less technically demanding than equilibrium dialysis[/U]. However, the solid phase purification and the GC-MS detection used by Van Uytfanghe et al. is cumbersome and time-consuming which makes it difficult for routine clinical testing.[/B] Previously we reported an LC-MS/MS procedure [4] for the measurement of serum total testosterone.[B] Here we describe a [U]new method using UF coupled with our testosterone LCMS/MS procedure for the measurement of FT offering further improvements in analytical sensitivity, convenience, and decreased sample requirement[/U]. A split sample comparison against analog immunoassay, cFT, and ED coupled LC-MS/MS is also presented.[/B][/I] [B]Discussion[/B][I] Reliable measurement of free steroid and thyroid hormones in the blood is inherently technically challenging and until recently has been difficult to achieve in routine clinical practice. [B]However, the increased sensitivity and ease of use of LC-MS/MS technology has made this an attractive, if not superior, alternative to immunoassay, and when coupled to prior ultrafiltration (UF) or equilibrium dialysis (ED) to remove the protein-bound fraction, a powerful tool to measure free hormone and drug levels.[/B] Several methods using just this technology have recently been published for the measure of free thyroid hormone and unbound antiretroviral drugs [23-25]. [B]Although both UF and ED are acceptable as reference procedures to separate protein-bound from circulating free ligands, [U]UF is inherently better suited to the demands of the clinical lab because of its greater simplicity and speed, and accordingly, we chose to implement it in our proposed method[/U]. [U]To the best of our knowledge, our proposed method is the first in the literature that directly measures FT by LC-MS/MS[/U]. *The UF-LC-MS/MS method described here compares favorably to the UF coupled GC-MS reference method recently published by Van Uytfanghe et al. [16]. This method achieves equivalent LLOQ (16 pmol/L vs. 15–20 pmol/L) with less sample (0.5 mL vs. 1 mL serum), higher throughput (3.9 vs. 10.45 min LC elution), and no need for derivitization. The Centrifree® ultrafiltration device is designed for the rapid separation of free ligands by the unhindered passage through the YM hydrophilic and nonabsorptive membrane (30 kDa cutoff) with a high degree of protein retention. [U]This membrane was ideal in that it truly retained the protein present in the serum (less than 0.07 g/L, the limit of detection of our protein assay, in the ultrafiltrate) while not adsorbing FT (Table 1, Supplementary Data)[/U]. Of note, the Centrifree® ultrafiltration device only worked when spun in a fixed angle rotor; swinging bucket rotors were unsuitable. [U]Our proposed method is attractive in that it agrees closely with ED and LC-MS/MS which must be considered the gold standard, but is much faster and easier to run, and better suited for the routine clinical laboratory, than the latter[/U]. [U]The ultrafiltration procedure requires approximately 1 h to complete while equilibrium dialysis requires at least 16 h to complete the dialysis step[/U]. The dynamic range of the procedure described here easily accommodates FT levels expected in the adult male, and lipemia, icterus, and other steroids do not interfere, and imprecision is less than 6% at FT levels expected in the adult male.[/B] The limitation of the current LLOQ makes the method is not sufficient for all female samples at low levels. However, for female test application, it is the high end of the range most clinical interested, i.e. hyperandrogenic conditions such as polycystic ovary syndrome. The 16 pmol/L LLOQ needs to be reduced by at least half to make the method suitable to measure with the imprecision of less than 20% of the FT levels expected in many women. Reducing the functional sensitivity by 50% might be achieved by increasing the serum ultrafiltrate from 0.5 to 1 mL or by adopting atmospheric pressure photoionization, but these will require additional studies to substantiate.[B] T[U]he overall fraction of total testosterone concentration measured by the present UF-LC/MS/MS method is 1.72%, which is very close to the reported before as 1.87% by UFGC/MS[/U] [17]. Further clinical studies are needed for the evaluation of clinical utilities of FT by this UF coupled LC-MS/MS method in subpopulations such as hypoandrogenic men and polycystic ovary syndrome women.[/B][/I] [B]*[I]In conclusion, we have developed a simple, rapid, highly selective, and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput, and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice.[/I][/B] [ATTACH type="full" alt="Screenshot (10559).png"]19270[/ATTACH] [ATTACH type="full" alt="Screenshot (10560).png"]19271[/ATTACH] [B]Fig. 4. Method comparison of UF coupled LC-MS/MS with ED coupled LC-MS/MS (n= 26). The upper panel shows the correlation plot of UF vs. ED. The lower panel shows plots of the bias between free testosterone measurements by UF and ED as a percentage of ED values (Y-axis) against the ED measured FT concentration. UF, ultrafiltration coupled LC-MS/MS; ED, equilibrium dialysis coupled LC-MS/MS.[/B] [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
LabCorp Vs Quest free testosterone lab ranges: Why are they different?
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