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Development of a UPLC-MS/MS Method for the Determination of Norethindrone from Human Plasma Lan Li, Jinlan Dong, Tracey Paxon, Lin Zhang, Yuan-Shek Chen, Ben Hsu QPS, LLC, Newark, Delaware Norethindrone is a form of progesterone, a female hormone. It binds to the progesterone intracellular receptors in the reproductive system to inhibit ovulation and change the normal cycle of cervical mucus and endometrium. For these reasons, norethindrone is used to prevent pregnancy, to treat menstrual disorders, endometriosis, and other diseases caused by a hormone imbalance. Due to a lack of easily ionized structure moiety for mass spectrometry, quantification of norethindrone at mid- picogram level is challenging, and analytical methods often require large sample volume and complicated sample processing procedures. In our approach, the MS sensitivity of norethindrone was enhanced by utilizing a low cost derivatization agent. With a one-step liquid-liquid extraction on Tomtec, we achieved a lower limit of quantification (LLOQ) of 50 pg/mL, with 50 μL of sample consumption. 1. We successfully developed a low-cost derivatization method for Norethindrone utilizing Hydroxylamine to increase the MS sensitivity to achieve an LLOQ at 50 pg/mL. 2. With the one-step Liquid-Liquid extraction on Tomtec and derivatization, a LC-MS/MS method was successfully validated for Norethindrone in human plasma at an assay range of 50 to 10,000 pg/mL. Compound DP (V) CE (eV) CXP (V) Transition (m/z) Norethindrone 50 40 15 314.2→124.2 Norethindrone-d 6 50 40 15 320.2→128.2 HPLC: Shimadzu Nexera MS/MS: AB Sciex API-6500 Column: Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 µm, Waters Mobile Phases: A: Water:Acetonitrile:Acetic Acid @ 65:35:0.1 (v:v:v) B: Acetonitrile Solvent Program: Isocratic MS Parameters: Analyte LLOQ QC L, M and HQC Intra-run Inter-Run Intra-run Inter-Run Norethindrone %CV 2.9 to 5.2 4.7 0.7 to 5.3 2.0 to 3.8 %RE -8.2 to -2.6 -4.6 -9.6 to -3.5 -8.4 to -6.0 Precision and Accuracy Linearity Assay Range: 0.05-10 ng/mL Linearity: 0.0918119754 Intercept: -0.000660934 R 2 : 0.9968 Possessed Sample Stability at 4 o C Time (hours) Concentrations (ng/mL) 0.150 (LQC) 8.00 (HQC) 123 0.171 8.54 0.171 8.49 0.176 8.52 0.163 8.41 0.159 8.59 0.157 8.46 n 6 6 Mean 0.166 8.50 S.D. 0.00760 0.0631 %CV 4.6 0.7 %RE 10.7 6.3 Lot Number Spike-In Concentration 0.0500 ng/mL %RE Lot 1 0.0450 -10.0 Lot 2 0.0478 -4.4 Lot 3 0.0465 -7.0 Lot 4 0.0499 -0.2 Lot 5 0.0491 -1.8 Lot 6 0.0435 -13.0 Mean 0.0470 S.D. 0.00245 %CV 5.2 %RE -6.0 n 6 Spike-In Selectivity Recovery and Matrix Factor Concentrations (Analyte/IS) 0.150/2.50 ng/mL 1.00/2.50 ng/mL 8.00/2.50 ng/mL % Recovery 80.3 86.6 87.5 Mean % Recovery 84.8 IS Normalized Matrix Factor 0.986 NA 0.978 Introduction Methods Summary Chromatograms LC-MS/MS Conditions Norethindrone 50 μL sample, 25 μL IS Liquid-liquid Extraction on Tomtec Dry Down @ 40 o C Reconstitution and Derivatization @ Ambient Temperature LC-MS/MS Analysis No extra steps !! Norethindrone C 20 H 26 O 2 MW: 298.42 Derivatized Norethindrone C 20 H 27 NO 2 MW: 313.43 MRM: m/z 314.2 [M+H] + m/z 124.2 124.2 Hydroxylamine NH 2 OH MW: 33.03 Derivatization Results I n t e n s i t y LLOQ Extracted Blank IS Time (min)

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  • Development of a UPLC-MS/MS Method for the Determination of Norethindrone from Human Plasma

    Lan Li, Jinlan Dong, Tracey Paxon, Lin Zhang, Yuan-Shek Chen, Ben HsuQPS, LLC, Newark, Delaware

    Norethindrone is a form of progesterone, a female hormone. It binds to theprogesterone intracellular receptors in the reproductive system to inhibitovulation and change the normal cycle of cervical mucus and

    endometrium. For these reasons, norethindrone is used to preventpregnancy, to treat menstrual disorders, endometriosis, and other diseasescaused by a hormone imbalance. Due to a lack of easily ionized structuremoiety for mass spectrometry, quantification of norethindrone at mid-picogram level is challenging, and analytical methods often require largesample volume and complicated sample processing procedures. In ourapproach, the MS sensitivity of norethindrone was enhanced by utilizing alow cost derivatization agent. With a one-step liquid-liquid extraction on

    Tomtec, we achieved a lower limit of quantification (LLOQ) of 50 pg/mL,with 50 μL of sample consumption.

    1. We successfully developed a low-cost derivatization method for Norethindrone utilizingHydroxylamine to increase the MS sensitivity to achieve an LLOQ at 50 pg/mL.

    2. With the one-step Liquid-Liquid extraction on Tomtec and derivatization, a LC-MS/MSmethod was successfully validated for Norethindrone in human plasma at an assayrange of 50 to 10,000 pg/mL.

    Compound DP (V) CE (eV) CXP (V) Transition (m/z)

    Norethindrone 50 40 15 314.2→124.2

    Norethindrone-d6 50 40 15 320.2→128.2

    HPLC: Shimadzu NexeraMS/MS: AB Sciex API-6500Column: Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 µm, WatersMobile Phases: A: Water:Acetonitrile:Acetic Acid @ 65:35:0.1 (v:v:v)

    B: AcetonitrileSolvent Program: Isocratic

    MS Parameters:

    AnalyteLLOQ QC L, M and HQC

    Intra-run Inter-Run Intra-run Inter-Run

    Norethindrone%CV 2.9 to 5.2 4.7 0.7 to 5.3 2.0 to 3.8

    %RE -8.2 to -2.6 -4.6 -9.6 to -3.5 -8.4 to -6.0

    Precision and Accuracy

    Linearity

    Assay Range: 0.05-10 ng/mLLinearity: 0.0918119754Intercept: -0.000660934R2 : 0.9968

    Possessed Sample Stability at 4oC

    Time (hours)

    Concentrations (ng/mL)

    0.150 (LQC) 8.00 (HQC)

    123

    0.171 8.54

    0.171 8.49

    0.176 8.52

    0.163 8.41

    0.159 8.59

    0.157 8.46

    n 6 6

    Mean 0.166 8.50

    S.D. 0.00760 0.0631

    %CV 4.6 0.7

    %RE 10.7 6.3

    Lot Number

    Spike-In Concentration0.0500 ng/mL

    %RE

    Lot 1 0.0450 -10.0

    Lot 2 0.0478 -4.4

    Lot 3 0.0465 -7.0

    Lot 4 0.0499 -0.2

    Lot 5 0.0491 -1.8

    Lot 6 0.0435 -13.0

    Mean 0.0470

    S.D. 0.00245

    %CV 5.2

    %RE -6.0

    n 6

    Spike-In Selectivity

    Recovery and Matrix Factor

    Concentrations (Analyte/IS)

    0.150/2.50 ng/mL 1.00/2.50 ng/mL 8.00/2.50 ng/mL

    % Recovery 80.3 86.6 87.5

    Mean % Recovery 84.8

    IS Normalized Matrix Factor

    0.986 NA 0.978

    Introduction

    Methods

    Summary

    Chromatograms

    LC-MS/MS Conditions

    Norethindrone

    50 µL sample, 25 µL IS

    Liquid-liquid Extraction on Tomtec

    Dry Down @ 40oC

    Reconstitution andDerivatization @ Ambient

    Temperature

    LC-MS/MS Analysis

    No extra steps !!

    Norethindrone

    C20H26O2MW: 298.42

    Derivatized Norethindrone

    C20H27NO2MW: 313.43

    MRM: m/z 314.2 [M+H]+ m/z 124.2

    124.2

    Hydroxylamine

    NH2OHMW: 33.03

    Derivatization Results

    Int

    ensity

    LLOQ

    Extracted Blank

    IS

    Time (min)


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