23


result, GC-MS/MS provides the specificity, sensitivity and precision required to reliably interpret physiologically relevant levels of free testosterone in plasma.


An experiment was developed to demonstrate the high sensitivity, precision and accuracy offered by GC-MS/MS. A bioanalytical method was used for the analysis of free testosterone at physiologically relevant levels in plasma ultra- filtrate (UF) from post-menopausal women.


Experimental EDTA (ethylenediaminetetraacetic acid) plasma from post-menopausal women was stored at -20°C and used to form quality control samples (QCs). The plasma was also stripped with charcoal to prepare calibration standards (STDs). Plasma UF was prepared using a centrifugal filter device (Centricon YM- 30, Millipore Corporation, Billerica, MA). Sample preparation was completed in three steps. First, a penta deuterated testosterone (d5- testosterone) solution was added to 0.5 mL of the plasma UF, followed by liquid:liquid hexane/ethyl acetate extraction. Oxime of the steroids was then prepared by evaporating a volatile extraction solvent with N2 and derivatizing with O-(2,3,4,5,6 pentafluorobenzyl)-hydroxylamine HCl. Silylation of the OH group was enabled using N-Methyl-N-trifluoroacetamide (MSTFA).


A gas chromatograph (Varian 3400, Varian, Inc., Walnut Creek, CA) was implemented together with a 15 m x 0.250 mm DB-1 fused silica capillary column (J&W Scientific, Folsom, CA). The split flow rate was 50:1 and the temperature program spanned from 210°C to 303°C. A triple stage mass spectrometer (Finnigan TSQ-7000, Thermo Fisher Scientific, San Jose, CA) was also used, operated in negative ion chemical ionization (NICI) mode.


Results and Discussion The assay range of the GC-MS/MS method was determined to be between 0.250 and approximately 100 pg/mL. The calibration standard curves yielded consistent regression slopes and all acceptance criteria were met. Table 1 demonstrates the intrarun and interrun precision and accuracy results whereas Figure 1 shows the chromatograms that were derived over the course of the experiment. Recovery was 78-79%. UF samples were stable after three freeze/thaw cycles, after 30.5 hours at ambient temperature, after 42 days at -20°C and after 46 days at -70°C. Extracts were stable in the autosampler after 239 hours. Testosterone levels were stable in human plasma stored at -70°C for at least 62 days and -20°C for at least 42 days.


Run 1 Mean CV (%)


Bias (%)


Run 2 Mean CV (%)


LLOQ


Low


Medium


ULOQ


0.250 pg/mL 1.29 pg/mL 9.56 pg/mL 96.1 pg/mL Intrarun


0.242 14.8 -3.2


1.37 6.69 6.20


N5 6 0.222


Bias (%)


Run 3 Mean CV (%)


10.4 -11.2


N6 6 0.207


Bias (%)


22.5 -17.2


N5 5 Interrun


Mean CV (%)


Bias (%) N


0.224 16.1 -10.4 16


1.29 7.16 0.00 17


9.56 4.36 0.00 18


Table 1. Intrarun/Interrun Precision and Accuracy. LLOQ is the the lower limit of quantitation and ULOQ is the upper limit of quantitation.


96.1 4.66 0.00 18


1.25 3.26 -3.10


1.25 7.00 -3.10


9.36 7.18 -2.09 6


9.61 2.26


0.523 6


9.73


0.741 1.78 6


94.9 4.58 -1.25 6


96.1 6.50 0.00 6


97.3 2.70 1.25 6


a) blank


b) LLOQ (0.25 pg/mL)


c) ULOQ (100 pg/mL)


Note: in each panel, the first chromatogram is a trace of the IS ion channel; the second is a trace of the ion channel for testosterone. The second peak in the LLOQ and ULOQ chromatograms is due to the testosterone isomer formed during derivatization. Figure 1. Chromatograms of Extracted Standards that are: a) blank, b) at the LLOQ, and c) at the ULOQ


GC-MS/MS was able to routinely and reproducibly deliver results at a LOQ of 3 femtograms on column, considerably lower than most LC-MS/MS systems can routinely deliver. The liquid-liquid extraction technique used for this assay is proven to reduce matrix effects, resulting in the analytical column always being exposed to the cleanest possible biological samples. As a result, build-up of problematic matrix components such as phospholipids, polar metabolites and other polar interferences was reduced. (8) In addition, the derivatization step required to make compounds volatile for injection into the GC offered some degree of specificity.


The GC column provided increased efficiency of chromatographic separation, generating highly focused peaks. Since the


majority of the analyte was chromatographically concentrated in a narrow peak (3 seconds or less at base), separation from background noise became easier, resulting in clear delineation as to when the signal started and ended. Consequently, precision was improved since peak inflection and deflection points were easier to assess through automated peak integration algorithms.


The entire column effluent from the GC column entered the MS ion source making it far more efficient than atmospheric pressure ionization (API) techniques employed by LC- MS/MS. API sources (ESI/APCI) are usually 1- 10% efficient in transferring the ions from the LC eluent into the MS. This means while close to 100% of the column eluent enters


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