LC/MS/MS Determination Of Leuprolide In Human Plasma

Presented by Rand Jenkins and Scott Wright PPD Development

Introduction
A bioanalytical method was developed for the analysis of leuprolide and its isotopically labeled internal standard (leuprolide-13C6,15N), from human plasma, containing tripotassium EDTA, by solid phase extraction using ISOLUTE 100mg C18 (EC) SPE cartridges.

Introduction
Analysis was performed on a SCIEX API 4000 triple quadrupole mass spectrometer using a Turbo IonSpray interface. Positive ions, formed by protonation of the analyte molecules, were measured using multiple reaction monitoring (MRM) mode. The total run time was approximately 5.0 minutes.

Introduction
The assay was validated over the concentration range of 0.0250 to 25.0 ng/mL and requires a 500-L human plasma aliquot. Samples are kept frozen at -20 C prior to analysis. A validation was performed to evaluate the specificity, precision, accuracy, recovery, and stability characteristics of the assay. The validation data presented demonstrate that the assay meets the performance requirements needed to support its use for clinical studies.

Objective
To develop and validate a rapid, selective, and sensitive bioanalytical assay for the determination of leuprolide in human plasma. Figure 1. Chemical Structure Information
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Summary Of Bioanalytical Method

Validation Samples
Calibration standards containing leuprolide were prepared in human plasma (tripotassium EDTA) at ten concentration levels spanning the quantitation range of 0.0250 to 25.0 ng/mL. Quality control (QC) pools were prepared in human plasma at the lower limit of quantitation and three additional concentrations spanning the quantitation range for the evaluation of assay performance, stability, and recovery.

Extraction Method
A 500-mL sample aliquot was fortified with 25 mL of internal standard working solution and diluted with 500 mL of phosphate buffer, pH 7. Analytes were isolated through solid phase extraction using ISOLUTE 100-mg C18 (EC) SPE cartridges preconditioned with 3 x 350 mL of methanol and 3 x 350 mL of water.

Extraction Method
The extraction was automated using the Tomtec Quadra96 automated workstation. The extraction plate was washed with 3 x 350 mL each of water and then 0.1% formic acid in 90:10 water/acetonitrile. The analytes were eluted from the SPE packing with 3 x 350 mL of methanol. The eluate was evaporated; the remaining residue was reconstituted with 150 mL of 50:50 methanol/water.

Chromatographic and Mass Spectrometric Conditions

The extracted samples (25 mL) were injected onto a Develosil C30, RP-Aqueous 2.0 mm x 150 mm, 5 mm, analytical column connected to an LC/MS/MS system. Mobile Phase A was 0.1% Formic Acid in 5.0 mM Ammonium Formate; Mobile Phase B was 0.1% Formic Acid in 60:40 Acetonitrile / 5.0 mM Ammonium Formate, v/v. Gradient program:
Time (min) 0.0 1.2 2.0 2.01 4.5 Flow Rate (mL/min) 0.300 0.300 0.300 0.300 0.300 % MP B 50 95 95 50 50

Chromatographic and Mass Spectrometric Conditions

The analytes were detected by multiple reaction monitoring on a SCIEX API 4000 triple quadrupole mass spectrometer system using positive ion Turbo IonSpray. Retention times for leuprolide and its internal standard were approximately 1.8 min.

Calculations
Calibration curves were constructed using the chromatographic peak area ratios of the analyte and internal standard from the calibration samples by applying a linear, weighted 1/concentration squared (1/c2) regression algorithm. Analyte concentrations in unknown samples were then calculated from their peak area ratios versus the calibration curve.

Results
The assay was validated over three separate validation runs. The results of the validation are summarized in the following tables.

Results
Table 1. Regression Equations and Correlation Coefficients
Regression Equation Y = 2.366307E Y = 2.687455E Y = 2.590604E - 03 + 6.834584E - 03 + 6.738148E - 03 + 7.933243E - 01 * X - 01 * X - 01 * X R 0.9987 0.9995 0.9985 0.9989

Average Correlation Coefficient

Results
Table 2. Intra-Assay Precision and Accuracy
Sample IA LOQ IA 1 IA 2 IA 3 %CV 7.66 2.80 2.13 2.08 %Bias -10.1 -4.85 -0.877 -0.710 n 6 6 6 6

Results
Table 3. Inter-Assay Precision and Accuracy
Sample QC LOQ QC 1 QC 2 QC 3 %CV 10.1 3.98 2.20 1.92 %Bias -8.19 -3.22 -2.20 -1.16 n 18 18 18 18

Results
Table 4. Stability Summary
Stability Conditions Freeze/Thaw Stability Extract Stability Matrix Stability Minimum Stability 3 Freeze/Thaw Cycles 117 Hours at Room Temperature 27 Hours at Room Temperature

Results
Figure 2. Plasma Blank with Internal Standard

Results
Figure 3. Lower Limit of Quantitation, 0.0250 ng/mL

Conclusions
An LC/MS/MS assay has been successfully developed and validated for the determination of leuprolide in human plasma containing tripotassium EDTA. The assay is suitable for the analysis of clinical study samples as demonstrated by its specificity, precision, accuracy, recovery, and stability characteristics.

Acknowledgements
The authors would like to acknowledge Margaret L. Wares contribution to the poster presentation.