A highly complex, sensitive and simultaneous LC-MS/MS method for the quantification of Total Estrone & Total Equilin in human plasma

Estrone is one of the major naturally occurring (endogenous) steroidal estrogens, while Equilin is classified as a non-endogenous estrogen. They are used for the management of perimenopausal and postmenopausal symptoms.

Study Design



Since both molecules have their in vivo metabolites, namely sulfate and glucuronide, there is a regulatory requirement to establish a bioanalytical method for assessing both the free and conjugated components (i.e., total Estrone and total Equilin) in clinical study samples. At Lambda Therapeutic Research, we have developed and validated a highly sensitive bioanalytical method capable of quantifying total Estrone and total Equilin in a single analytical run.


Developing a method for quantifying low-dosage, endogenous Estrone and non-endogenous Equilin required innovative approaches to handle interference-free matrix preparation, efficient enzymatic conversion, and matrix effect mitigation, while also addressing derivatization needs and potential interference from structurally similar compounds in the biological matrix.

  • Both molecules belong to the families of steroids/hormones, and their dosages are extremely low, resulting in detection levels in the low picogram range. Since Estrone is an endogenous compound, the need for a surrogate matrix to create an interference-free matrix for the preparation of CC/QC samples arises. Hence, a charcoal stripping method was developed to prepare interference-free human plasma. On the other hand, Equilin is a non-endogenous compound, and developing a single-run method for Equilin posed challenges in minimizing matrix effects between the surrogate and actual matrix.
  • Due to the requirement for developing a method for total Estrone and Equilin (as conjugated forms), it was necessary to convert sulfate and glucuronide metabolites into their respective parent molecules. To achieve this, three different types of enzymes were selected and evaluated. One enzyme was chosen that exhibited efficient sulfatase and glucuronidase activity in the actual matrix. A unique set of conditions involving incubation time, temperature, and buffer solution addition for sample preparation was optimized to ensure proper conversion and accuracy.
  • Given the widely accepted practice to ionize steroids/hormones in triple quadrupole mass spectrometry, derivatization becomes essential for achieving low picogram levels in a biological matrix. Hence, after enzymatic reaction and sample cleanup using solid-phase extraction, a derivatization reaction was implemented prior to LC-MS/MS analysis.
  • Due to the presence of structurally similar compounds and related hormones in the formulation and biological matrix, the potential for interference with targeted analyte chromatography was significant. To address this challenge, we employed a unique core-shell technology-based analytical column.

At Lambda, we have developed a multi-step and robust method encompassing enzymatic reaction, solid-phase extraction, derivatization, and liquid-liquid extraction. This comprehensive approach ensures both acceptable precision and accuracy while effectively addressing matrix effect-related concerns.

Extraction Method flow

Chromatography of LLOQ sample




Below are the global statistics for three precision and accuracy runs conducted on both altered and unaltered matrices.

Equilin – Global Statistics (Altered matrix QCs)
Mean (pg/mL)7791.43684357.4172118.558240.4250
Precision (%CV)
Nominal value(pg/mL)7952.3464373.791119.71141.500
Accuracy %98.099.699.097.4
Estrone – Global Statistics (Altered matrix QCs)
Mean (pg/mL)7564.24544315.3342116.271143.7111
Precision (%CV)
Nominal value(pg/mL)7950.9934373.046119.69041.493
Accuracy %95.198.797.1105.3
Equilin – Global Statistics (Un-altered matrix QCs)
Mean (pg/mL)7059.69583949.43331009.5493116.247335.6806
Precision (%CV)
Nominal value (pg/mL)7930.9914481.0091053.037118.79540.391
Accuracy %
Estrone – Global Statistics (Un-altered matrix QCs)
Mean (pg/mL)7383.56714180.98371145.2358107.272147.1621
Precision (%CV)
Nominal value (pg/mL)8012.4944580.0411169.486107.69744.662
Accuracy %92.291.397.999.6105.6

We have generated matrix stability data, including benchtop stability for 13.0 hours, freeze-thaw stability for five cycles, and long-term stability in matrix for 81 days as part of the method validation process.


The validated method has been successfully applied to analyze study samples in two different studies, in which incurred sample reproducibility experiment was conducted. The results were found to be well within the acceptable criteria for both analytes, thus demonstrating the method’s reproducibility for incurred samples.


A selective, precise, accurate, simultaneous, and highly sensitive LC-MS/MS method is now available for the quantification of Total Estrone and Total Equilin in human plasma.