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* A standardized ED method is recommended to effectively separate FT from the serum matrix.
* With the rapid development of liquid chromatography-tandem mass spectrometry (LC-MS/MS), the ED separation combined with LC-MS/MS has become the “gold standard” for quantifying FT [4].
* Serum FT concentration can be as low as 1% of serum TT [15]. The lower limit of female serum TT reference interval could be as lowas 0.29 nmol/L [14]. Thus, to accommodate measuring female serum FT, the LLMI for an LCMS/MS method would ideally be lower than 3 pmol/L. Therefore, a rapid and sensitive ED LC-MS/MS method for FT is required for clinical use.
Abstract
Objective
To compare the calculated and the rapid equilibrium dialyzed (ED) human serum free testosterone (FT) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to explore their performances in polycystic ovary syndrome (PCOS) diagnosis.
Methods
A rapid ED LC-MS/MS method for serum FT was established and validated forl inearity, lower limit of the measuring interval (LLMD, imprecision, trueness, stability, dilution, matrix specificity and carryover. The validated ED LC-MS/MS (ED-FT) was compared with calculated LC-MS/MS method from Vermeulen’s formula (cFT) for FT measurement in 139 PCOS patients and 100 healthy controls. The performances of total testosterone (TT), ED-FT and cFT by LC-MS/MS in PCOS diagnosis were investigated with the same cohorts.
Results
The linearity range of ED-FT was 1.74 - 890 pmol/L, with a LLMI at 1.74 pmol/L. The intra-assay and inter-assay imprecision were < 3.8% and < 5.9%. The trueness was acceptable with recoveries of 92.9% - 108.2%. The equilibrium dialysis time was 4 h. The two FT methods displayed systematic and proportional differences and cFT showed significant positive deviations compared to ED-FT. Receiver operating characteristic curve analysis proved that ED-FT out performanced in PCOS diagnosis with the an area under the curve at 0.973, sensitivity of 89.93% and specificity of 96.00%.
Conclusions
This study established a rapid, accurate and sensitive ED LC-MS/MS method for serum FT. ED-FT is optimal compared to TT and cFT by LC-MS/MS in PCOS diagnosis.
1 Introduction
Testosterone, an androgen, plays a vital role after puberty in spermatogenesis and follicle development, regulates muscle development and fat distribution, and red blood cell production[1]. In females, testosterone is primarily produced and secreted by adrenal gland and ovaries.The testosterone levels in polycystic ovarian syndrome (PCOS) patients are usually elevated compared to healthy females, resulting in endocrine and reproductive problems [2].
Most circulating testosterone is tightly bound to sex hormone-binding globulin (SHBG) or loosely bound to albumin (ALB), orosomucoid, and corticosteroid-binding globulin. Merely 1-4% of circulating testosterone is unbound or free. Only unbound or free testosterone (FT) couldenter the cell to exert biological effects [3]. Total testosterone (TT) is the sum of the bound and FT concentrations in the circulation. It is susceptible to changes in SHBG concentrations under the influence of gender, age, weight, metabolic syndrome, polymorphisms in the SHBG gene, and some medications [4]. Since FT is not affected by SHBG, it is an ideal indicator to assess the biologically active testosterone status. The accurate measurement of circulating FT is crucial and has been used in PCOS [5] diagnosis.
The extremely low serum FT concentration poses significant challenges for measurement techniques. Currently, most laboratories indirectly determine serum FT concentrations by calculating different mathematical models using TT, SHBG, and ALB (cFT) concentrations[6]. However, there were significant variations between the results of the various models. The direct serum FT measurement methods include two processes: separation of FT from the serum matrix and quantification of the separated FT. The techniques to separate FT from the serum matrix include equilibrium dialysis (ED) [7,8], ultrafiltration [9], and direct tracer analog immunoassays [10]. However, the direct tracer analog method should be avoided due to its inaccuracy [11]. The problem of nonspecific binding during ultrafiltration would also affect the accuracy [12]. A standardized ED method is recommended to effectively separate FT from the serum matrix. Quantification of FT in the dialysate was previously measured byimmunoassays. However, immunoassays suffer from poor specificity due to cross-reactions with other structurally similar steroids [13]. With the rapid development of liquid chromatography-tandem mass spectrometry (LC-MS/MS), the ED separation combined with LC-MS/MS has become the “gold standard” for quantifying FT [4].
Recent published ED LC-MS/MS methods for serum FT [7-10, 14] could be further improved. In these methods, the time required for the ED process ranged from 5 to 24 hours, and the lower limit of measuring interval (LLMJ) ranged from 2.4 to 16 pmol/L. Lengthy ED time affects the turnaround time for result reporting. Serum FT concentration can be as low as 1% of serum TT [15]. The lower limit of female serum TT reference interval could be as lowas 0.29 nmol/L [14]. Thus, to accommodate measuring female serum FT, the LLMI for an LCMS/MS method would ideally be lower than 3 pmol/L. Therefore, a rapid and sensitive EDLC-MS/MS method for FT is required for clinical use.
This paper reports the development and validation of an ED LC-MS/MS serum FT method with improved ED procedure and sufficient sensitivity to accommodate female samples. The improved method was evaluated for its clinical performance in diagnosing PCOS and compared with the performance of TT and cFT.
Figure 1. Comparison between cFT by LC-MS/MS and ED-FT by LC-MS/MS in 239 total subjects, 100 healthy controls as well as 139 PCOS patients. Passing-Bablok regressions (A) and bland-Altman plots (B). Red dots represent PCOS patients samples (n=139) and blue dots represent healthy controls samples (n=100).
* With the rapid development of liquid chromatography-tandem mass spectrometry (LC-MS/MS), the ED separation combined with LC-MS/MS has become the “gold standard” for quantifying FT [4].
* Serum FT concentration can be as low as 1% of serum TT [15]. The lower limit of female serum TT reference interval could be as lowas 0.29 nmol/L [14]. Thus, to accommodate measuring female serum FT, the LLMI for an LCMS/MS method would ideally be lower than 3 pmol/L. Therefore, a rapid and sensitive ED LC-MS/MS method for FT is required for clinical use.
Abstract
Objective
To compare the calculated and the rapid equilibrium dialyzed (ED) human serum free testosterone (FT) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to explore their performances in polycystic ovary syndrome (PCOS) diagnosis.
Methods
A rapid ED LC-MS/MS method for serum FT was established and validated forl inearity, lower limit of the measuring interval (LLMD, imprecision, trueness, stability, dilution, matrix specificity and carryover. The validated ED LC-MS/MS (ED-FT) was compared with calculated LC-MS/MS method from Vermeulen’s formula (cFT) for FT measurement in 139 PCOS patients and 100 healthy controls. The performances of total testosterone (TT), ED-FT and cFT by LC-MS/MS in PCOS diagnosis were investigated with the same cohorts.
Results
The linearity range of ED-FT was 1.74 - 890 pmol/L, with a LLMI at 1.74 pmol/L. The intra-assay and inter-assay imprecision were < 3.8% and < 5.9%. The trueness was acceptable with recoveries of 92.9% - 108.2%. The equilibrium dialysis time was 4 h. The two FT methods displayed systematic and proportional differences and cFT showed significant positive deviations compared to ED-FT. Receiver operating characteristic curve analysis proved that ED-FT out performanced in PCOS diagnosis with the an area under the curve at 0.973, sensitivity of 89.93% and specificity of 96.00%.
Conclusions
This study established a rapid, accurate and sensitive ED LC-MS/MS method for serum FT. ED-FT is optimal compared to TT and cFT by LC-MS/MS in PCOS diagnosis.
1 Introduction
Testosterone, an androgen, plays a vital role after puberty in spermatogenesis and follicle development, regulates muscle development and fat distribution, and red blood cell production[1]. In females, testosterone is primarily produced and secreted by adrenal gland and ovaries.The testosterone levels in polycystic ovarian syndrome (PCOS) patients are usually elevated compared to healthy females, resulting in endocrine and reproductive problems [2].
Most circulating testosterone is tightly bound to sex hormone-binding globulin (SHBG) or loosely bound to albumin (ALB), orosomucoid, and corticosteroid-binding globulin. Merely 1-4% of circulating testosterone is unbound or free. Only unbound or free testosterone (FT) couldenter the cell to exert biological effects [3]. Total testosterone (TT) is the sum of the bound and FT concentrations in the circulation. It is susceptible to changes in SHBG concentrations under the influence of gender, age, weight, metabolic syndrome, polymorphisms in the SHBG gene, and some medications [4]. Since FT is not affected by SHBG, it is an ideal indicator to assess the biologically active testosterone status. The accurate measurement of circulating FT is crucial and has been used in PCOS [5] diagnosis.
The extremely low serum FT concentration poses significant challenges for measurement techniques. Currently, most laboratories indirectly determine serum FT concentrations by calculating different mathematical models using TT, SHBG, and ALB (cFT) concentrations[6]. However, there were significant variations between the results of the various models. The direct serum FT measurement methods include two processes: separation of FT from the serum matrix and quantification of the separated FT. The techniques to separate FT from the serum matrix include equilibrium dialysis (ED) [7,8], ultrafiltration [9], and direct tracer analog immunoassays [10]. However, the direct tracer analog method should be avoided due to its inaccuracy [11]. The problem of nonspecific binding during ultrafiltration would also affect the accuracy [12]. A standardized ED method is recommended to effectively separate FT from the serum matrix. Quantification of FT in the dialysate was previously measured byimmunoassays. However, immunoassays suffer from poor specificity due to cross-reactions with other structurally similar steroids [13]. With the rapid development of liquid chromatography-tandem mass spectrometry (LC-MS/MS), the ED separation combined with LC-MS/MS has become the “gold standard” for quantifying FT [4].
Recent published ED LC-MS/MS methods for serum FT [7-10, 14] could be further improved. In these methods, the time required for the ED process ranged from 5 to 24 hours, and the lower limit of measuring interval (LLMJ) ranged from 2.4 to 16 pmol/L. Lengthy ED time affects the turnaround time for result reporting. Serum FT concentration can be as low as 1% of serum TT [15]. The lower limit of female serum TT reference interval could be as lowas 0.29 nmol/L [14]. Thus, to accommodate measuring female serum FT, the LLMI for an LCMS/MS method would ideally be lower than 3 pmol/L. Therefore, a rapid and sensitive EDLC-MS/MS method for FT is required for clinical use.
This paper reports the development and validation of an ED LC-MS/MS serum FT method with improved ED procedure and sufficient sensitivity to accommodate female samples. The improved method was evaluated for its clinical performance in diagnosing PCOS and compared with the performance of TT and cFT.
Figure 1. Comparison between cFT by LC-MS/MS and ED-FT by LC-MS/MS in 239 total subjects, 100 healthy controls as well as 139 PCOS patients. Passing-Bablok regressions (A) and bland-Altman plots (B). Red dots represent PCOS patients samples (n=139) and blue dots represent healthy controls samples (n=100).
