5990-5877en.indd

LC/MS/MS qua ntitation of β-estradiol 17-acetate using an Agilent 6460 Triple Quadrupole LC/MS working in ESI negative ion mode Abstract
A sensitive, precise, and accurate quantitative liquid chromatography/tandem Agilent Technologies India Pvt. Ltd.
Bangalore India mass spectrometry (LC/MS/MS) method for the measurement of estradiol acetate in human plasma was developed. The sample preparation method employed solid phase extraction on Agilent SampliQ cartridges. The processed samples were chromatographed on an Agilent ZORBAX Eclipse Plus C18 analytical column and analyzed by triple-quadrupole tandem mass spectrometry in the multiple reaction monitoring (MRM) mode using negative electrospray ionization. The principal advantage of the LC/MS/MS method described in this application note is the simultaneous achievement of high absolute recovery (~ 80%), high sensi- tivity (LLOQ of 100 fg injected on-column or 28.6 pg/mL plasma), high precision (≤ 5.9% CV) and accuracy (81.5-118.1%), as well as excellent linearity over the concentration range 0.1 to 10000 pg/μL (R2 ≥ 0.998) with a short run time of Introduction
Experimental
β-Estradiol 17-acetate is a derivative of Chemicals
Figure 1. Highly sensitive analytical
Preparation of the stock solution, cali-
excellent separation of the analyte from bration and quality control standards
A 0.2 mg/mL stock solution of ß-estradiol 17-acetate was prepared in acetonitrile (ACN)/water, 50/50 v/v (diluent). To prepare the various cali- bration levels in aqueous solutions, the stock solution was diluted using the diluent at seven concentration linearity study were 0.1 pg/μL, 0.5 pg/ μL, 1 pg/μL, 10 pg/μL, 100 pg/μL, 1000 pg/μL, and 10000 pg/μL. In addition to the above concentration levels, a 0.2 pg/μL standard solution was also used for the analyte recovery Figure 1: β-Estradiol 17-acetate Calibration levels in plasma were prepared by spiking 100 µL of each standard solution to 250 µL of human plasma. A 500 µL aliquot of Milli-Q water was added followed by 150 µL of 0.5% formic acid. The samples were vortexed after each addition. Each sample was loaded onto SPE cartridges as described below. Three quality control (QC) samples were prepared at the following concentration levels to check for precision and accuracy: 20 pg/μL (low QC), 200 pg/μL (mid QC), and 2000 pg/μL (high QC).
Human plasma extraction procedure
Determination of extraction recovery
LC parameters
protocol is compiled in Figure 2.
The Agilent 1200 Series RRLC system comprised a well plate autosampler with sample thermostat, degasser, column compartment, and pump. A 0.12 mm inner diameter stainless steel capillary was used for all tubing connections. The tubing length was minimized to reduce peak dispersion. The analytical column used for this experiment was ZORBAX Eclipse Plus C18 RRHT 2.1 x 100 mm 1.8 micron 600 bar (part number: 959764-902); the temperature was 40°C. Solvent A was 0.05% ammonium hydroxide (NH OH) The pump delivered 80% B isocratic mobile phase at a flow rate of 0.5 mL/min. The run time was 1.2 min with no post run time. The flush port was acti-vated for 5 sec with ACN/water 80:20 v/v. The autosampler was set for a Figure 2: Solid Phase Extraction protocol used to extract ß-estradiol 17-acetate from human plasma. Triple-quadrupole parameters
Table 1 gives the compiled triple-
Data acquisition and processing
as the quantifier ion) and 58.9 Da (used Agilent MassHunter Workstation Data Acquisition software. Data processing was performed with Agilent MassHunter Quantitative Analysis Set value in 6460 Triple Quad
Parameter
(negative mode)
Table 1: Optimized triple-quadrupole parameters. Results and Discussion
Quantitation of ß-estradiol 17-acetate
in aqueous solutions
Figure 3 represents the precursor and
product ions of β-estradiol 17-acetate. For quantitation of β-estradiol 17-acetate, Figure 4 shows an overlay of a 100 fg
aqueous solution with a blank. 100 fg/μL standard solution yielded a S/N ratio of Figure 3: Fragmentation pattern for β-estradiol 17-acetate showing precursor and product ions at CE = 30 V. Figure 4: Overlay of a 100 fg β-estradiol 17-acetate chromatogram in aqueous solution (blue trace) with a blank (red trace). Quantifier - 7 Levels, 7 Levels Used, 28 Points, 28 Points Used, 0 QCs level). A calibration curve was constructed by plotting the peak area of β-estradiol 17-acetate versus concentration (0.1 pg/μL, 0.5 pg/μL, 1 pg/μL, 10 pg/μL, 100 pg/μL, fitted by 1/x weighting. The linearity of the relationship between peak area and β-estradiol 17-acetate concentration is demonstrated by a correlation coeffi-cient of R2 > 0.9999.
Figure 5: Linearity curve of β-estradiol 17-acetate from 0.1 pg to 10000 pg injected on-column linearity is shown in Figure 5 with an
excellent correlation coefficient of R2 > 0.9999. Quantitation of ß-estradiol 17-acetate
in human plasma
for potential interferences. The obvious Figure 6 shows an overlay of a 100 fg
S/N ratio > 38 (N = 4, Signal = Height, Figure 6: Overlay of a 100 fg β-estradiol 17-acetate chromatogram recovered from human plasma (blue trace) overlaid with a blank (red trace). Linearity, precision, accuracy, and
Quantifier - 8 Levels, 8 Levels Used, 32 Points, 32 Points Used, 12 QCs recovery
y = 1118.6141 * x + 87.0591R^2 = 0.99848795 Linearity was studied over the concentra-tion range of 100 fg/μL to 10000 pg/μL (eight levels, 4 replicates per level, 5 orders of magnitude). The correlation (Figure 7). The calculation was
regression (1/x). The results obtained
for linearity, precision, and accuracy
are shown in Table 2. The method
intra-day precision and accuracy values for low QC, mid QC, and high QC levels (20 pg/μL, 200 pg/μL, 2000 pg/μL, 85.0%, 103.2%, 100.9%, respectively. The injector precision and accuracy Figure 7: Linearity curve of β-estradiol 17-acetate recovered from human plasma from 0.1 pg to 10000 pg injected on-column (linear fit, weight 1/x). The blue triangles ( value of retention time at various linearity levels. Extraction recoveries of ß-estradiol 17-acetate from spiked plasma samples are found to be ~ 80% at 1 pg, 10 pg, and 100 pg concentration levels.
Linearity level
Retention time
Response RSD
Accuracy RSD [%)
amount on-column
response
accuracy
Table 2: RSD values for retention time, response, and accuracy from plasma linearity study (n = 4). Conclusions
• The linearity of the quantitation of LC/MS in ESI negative ion mode is demonstrated. on-column is demonstrated using Agilent SampliQ SPE cartridges. • The linearity of the quantitation of 10000 pg on-column). The observed R2 value for the calibration curve is > 0.9999.
This item is intended for Research Use Only. Notfor use in diagnostic procedures. Information,descriptions, and specifications in this publica-tion are subject to change without notice.
Agilent Technologies shall not be liable for errorscontained herein or for incidental or consequen-tial damages in connection with the furnishing,performance, or use of this material.
Agilent Technologies, Inc. 2010Published in the U.S.A. June 28, 20105990-5877 EN

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