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DEVELOPMENT OF A HIGHLY SENSITIVE LC-MS/MS METHOD
FOR THE SIMULTANEOUS DETERMINATION OF TESTOSTERONE
AND DIHYDROTESTOSTERONE IN HUMAN PLASMA
M. Boterman, F. Oostebring, M. Hogenberg and E. Oosting
Introduction
Materials and Methods
In humans, circulating androgens including testosterone (T) and its active
metabolite dihydrotestosterone (DHT) in plasma, exert major biological
effects at extremely low (namomolar or picomolar) concentrations. For
the measurement of T and DHT, most clinical and research laboratories
rely upon radioimmunoassays (RIAs) and more recently automated
immunoassays. However, other methods are becoming increasingly
available. Unfortunately, some of these methods lack precision and
specificity, particularly at low concentrations, making them unsuitable for
comparing androgen levels in women.
T, DHT and the internal standard d3-T, were extracted from plasma by a
liquid/liquid extraction with a mixture of pentane and diethylether. The
extracted samples were dried and subsequently subjected to a
derivatization with picolinic acid. Further sample cleanup was performed
with a solid phase extraction followed by a liquid/liquid extraction with
TBME. The extracted samples were introduced into the LC-MS/MS
system for quantification. The samples were chromatographed on a
Phenomenex Luna C18 column (3.5 µm, 100 x 2.0 mm). The massspectrometer consisted of a Sciex API 4000 equipped with a turbo ion
spray interface and was operating in the positive ion mode.
The objective of the present study was to develop and validate a highly
sensitive and reliable LC-MS/MS method for the determination of T and
DHT in human plasma. With a calibration range of 0.0500 – 20.0 ng/mL
this assay proved to be most adequate for the determination of T and
DHT in human male as well as female plasma.
Results
Calibration: The assay was validated in the concentration range of
0.0500 – 20.0 ng/mL for T and DHT in human heparin plasma. Since T
and DHT are both endogenous compounds, the calibrator samples were
not prepared in human heparin plasma. Instead, artificial matrix (4%
Bovine Serum Albumin in saline solution) was used as blank matrix. A
lower limit of quantification (LLOQ) of 0.0500 ng/mL was achieved for
both compounds (Figure 1 and Figure 2).
T
A full validation of the method was performed according to the current
guidelines for bioanalytical method validation[1,2]. The validation included
the determination of the parameters: calibration, accuracy and precision,
recovery, specificity, dilution, and stability.
Accuracy and precision: As the normal T and DHT levels in female
heparin plasma are markedly lower than in male plasma, Quality Control
samples were prepared in female heparin plasma. However, since the
QC-LLOQ and QC-Low levels for testosterone in this assay were below
or approximately at the normal endogenous levels in female plasma, both
the QC-LLOQ and QC-Low samples were prepared in artificial matrix.
The method showed acceptable accuracies (expressed as bias and
precisions (expressed as CV) for both compounds (Table 1 and Table 2).
Table 1. Accuracy and precision of testosterone in human heparin plasma
QC level
n
Mean (ng/mL)
Accuracy (%)
Bias (%)
Intra CV (%)
Total CV (%)
0.0500 ng/mL
LLOQ 1
23
0.0599
119.7
19.7
8.4
0.150 ng/mL
Low 1
23
0.160
106.7
6.7
6.0
1.69 ng/mL
Medium
24
1.72
101.5
1.5
1.3
15.2 ng/mL
High
24
15.6
102.3
2.3
1.2
150
ng/mL
OQC 2
18
153
102.1
2.1
1.5
1 LLOQ and QC-Low were prepared in artificial matrix
2 The “Over the curve” (OQC) samples were analyzed after a 10-fold dilution with artificial matrix.
11.1
7.7
2.5
1.7
1.6
Table 2. Accuracy and precision of dihydrotestosterone in human heparin plasma
QC level
Figure 1. LC-MS/MS chromatograms of T (upper panel) and d3-T (lower panel) at the LLOQ
DHT
n
Mean (ng/mL)
Accuracy (%)
Bias (%)
Intra CV (%)
Total CV (%)
0.0500 ng/mL
LLOQ 1
23
0.0570
114.1
14.1
5.6
0.150 ng/mL
Low 1
23
0.158
105.6
5.6
4.7
1.69 ng/mL
Medium
24
1.79
102.4
2.4
3.5
15.2 ng/mL
High
24
15.5
102.2
2.2
2.3
2
150
ng/mL
OQC
18
151
100.9
0.9
3.1
1 LLOQ and QC-Low were prepared in artificial matrix
2 The “Over the curve” (OQC) samples were analyzed after a 10-fold dilution with artificial matrix.
6.3
6.8
9.5
6.6
6.6
Recovery: The method was found valid with respect to recovery
(liquid/liquid extraction prior to derivatization).
Specificity: No influence was observed in six different sources of plasma
on spiked T and DHT concentrations at a low and high level. No relevant
interferences were observed at the retention time of the internal standard.
Dilution: The 10-fold dilution of plasma samples with blank artificial
matrix was considered valid.
Figure 2. LC-MS/MS chromatograms of DHT (upper panel) and d3-T (lower panel) at the LLOQ
Stability: 24h storage of samples on the bench and in the refrigerator,
69h storage of samples on the autosampler (at 10 ºC), three additional
freeze/thaw cycles and 66 days long-term storage at ≤-18 ºC were
successfully validated for both T and DHT.
Discussion and Conclusions
At ABL, a highly sensitive bioanalytical LC-MS/MS method for the simultaneous determination of T and DHT in human heparin plasma has been
developed and validated successfully. The method produced accurate and precise results and a very low LLOQ was achieved for both compounds.
Due to this low LLOQ, the assay has proved to be extremely suitable for quantitative bioanalysis, especially for measuring T and DHT levels in
women.
References
1 Viswanathan et al. Workshop/Converence Report – Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays. AAPS Journal 9
(2007) E30-E42.
2 Guidance for Industry, Bioanalytical Methods Validation. U.S. Department of Health and Human Services, FDA. June 2001.