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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 1  |  Page : 17-24

Development and validation of a liquid chromatography/tandem mass spectrometric method for determination of ethinyl estradiol in human plasma


1 Department of Pharmaceutical Chemistry, University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India
2 Analytical Sciences Research Area, Indian Institute of Chemical Technology, Hyderabad, Telangana, India

Date of Web Publication18-Oct-2017

Correspondence Address:
Vijay Kotra
Department of Pharmaceutical Chemistry, University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur - 522 510, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ddt.DDT_5_16

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  Abstract 

Introduction: A simple and rapid bioanalytical liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on solid phase extraction (SPE) followed by liquid-liquid extraction (LLE) has been developed and validated for quantification of Ethinyl Estradiol in human plasma. Methods: API 5500 LC-MS/MS system with turbo ion-spray interface equipped with pumps (Shimadzu LC-20ADVP), an auto Sampler (Shimadzu SIL-HTC), analytical column SB C18 HT (50 x 3.0 mm, 1.8 μ) and data acquisition system and quantitation program (Applied Biosystems Analyst Software version 1.5, Thermo scientific) was used. The Positive ions were measured in MRM mode for the analyte and Ethinyl Estradiol-d4 used as an internal standard. A composition of 2 mM ammonium formate buffer: acetonitrile (20: 80 v/v) was used as mobile phase. The total run time was 4.0 min. The proposed method has been validated with in the linear range of 5.000–308.560 pg/ml for Ethinyl Estradiol. Results: The retention times of Ethinyl Estradiol and Ethinyl estradiol-d4 were 3.42 min ± 0.30 min and 3.45 min ± 0.30 min respectively. The intraday and interday precision values were within 1.58% to 10.86% and 4.62% to 19.74% respectively for Ethinyl estradiol. The overall recovery for Ethinyl estradiol was found to be 86.91%-103.15%. Conclusion: The method was validated as per ICH guidelines and it would be useful for bioequivalence and pharmacokinetic studies of Ethinyl Estradiol in human plasma.

Keywords: Ethinyl estradiol, human plasma, LC-MS/MS, liquid–liquid extraction, solid-phase extraction


How to cite this article:
Kotra V, Ramisetti NR, Surapaneni R, Konidala SK. Development and validation of a liquid chromatography/tandem mass spectrometric method for determination of ethinyl estradiol in human plasma. Drug Dev Ther 2017;8:17-24

How to cite this URL:
Kotra V, Ramisetti NR, Surapaneni R, Konidala SK. Development and validation of a liquid chromatography/tandem mass spectrometric method for determination of ethinyl estradiol in human plasma. Drug Dev Ther [serial online] 2017 [cited 2017 Nov 17];8:17-24. Available from: http://www.ddtjournal.org/text.asp?2017/8/1/17/216932




  Introduction Top


Ethinyl estradiol (EE),[1] chemically known as 17 α-ethinyl-13-methyl-7, 8, 9, 11, 12, 14, 15, 16, 17-octahydro-6H-cyclopenta[a] phenanthrene-3,17-diol [Figure 1], is a semi-synthetic alkylated estradiol with a 17-α-ethinyl substitution.
Figure 1: Molecular structure of ethinyl estradiol

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It is an orally bio-active estrogen[2] used in almost all modern formulations of combined oral contraceptive pills. EE is absorbed with maximum plasma concentrations occurring within 2 h after drug administration. The absolute bioavailability of EE is about 43% and plasma protein binding is 97%. The Cmax and Tmax of EE are 102.4 pg/ml and 1.83 h, respectively.[3]

Literature survey reveals that only a few methods were reported for the quantification of EE in biological media. These include high-performance liquid chromatography (HPLC)[4],[5],[6],[7],[8],[9] and LC-MS/MS.[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20]

The present manuscript describes the development and validation of a simple and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method having analysis time of 4 min, sensitivity (5 pg/ml), utilizing small amount of plasma (100 μl plasma) for sample preparation, simple extraction, and analyte comparison with EE-d4 used as an internal standard (ISTD).


  Experiment Top


Chemicals and reagents

EE and EE-d4 were obtained from Famy care laboratories limited. HPLC grade formic acid (Merck), acetonitrile (J.T Baker), methanol (J.T Baker), ter-butyl methyl ether (Spectrochem) and acetone (Merck), ultra-pure ammonium formate (Fluka), and dansyl chloride (Fluka) were purchased from market. Eight lots of human K-EDTA plasma (blank matrix) were purchased. In-house prepared HPLC/Milli-Q water was used for analysis.

Instrumentation

API 5500 LC/MS/MS system equipped with pumps (Shimadzu LC-20ADvp), an auto sampler (Shimadzu SIL-HTc), analytical column SB C18 HT (50 mm × 3.0 mm, 1.8 μ), and data acquisition system and quantization program (Applied Biosystems Analyst Software Version 1.5) was used.

Preparation of standard solutions

The standard stock solution of EE (399.787 mg/ml) was prepared by dissolving its accurately weighed amount in methanol. The working solution was prepared by appropriate dilution of stock solution in diluent-1. Calibration standards (CSs) and quality control (QC) samples were prepared by spiking blank plasma with standard spiking solutions. CSs were prepared at 5.0, 10.0, 20.30, 60.90, 120.80, 182, 70, 243.60, and 308.56 pg/ml concentrations while QC samples were prepared at 179.520 pg/ml high QC (HQC), 89.760 pg/ml and 44.880 pg/ml (medium QC [MQC] M2QC and M1QC), 15.600 pg/ml low QC (LQC), and 5.200 pg/ml lower limit of quantification QC (LLOQ QC) concentrations. Stock solution (400.867 μg/ml) of the ion spray (IS) was prepared by dissolving 2.039 mg of d-4 EE in 5.0 ml of methanol. A working ISTD solution was prepared at 4.00 μg/ml concentration. Standard stock and working solutions used for spiking were stored at 5°C, while CSs and QC samples in plasma were kept at 70°C until use.

Preparation of samples

Solid-phase extraction

The analyte (EE) was extracted from plasma samples using a validated solid-phase extraction (SPE) technique. To a 400 μl volume of plasma aliquot (calibration sample or QC sample), 50 μl of ISTD working solution (EE-d4 10.000 ng/ml) was added except in blank sample. Add 300 μl of 0.2% formic acid in Milli-Q water. After vortex mixing for few seconds, complete contents (750 μl) were loaded onto a conditioned SPE cartridge (Oasis MCX Cartridges, 30 mg, 1cc). Load the samples and wash the SPE cartridge with 1 ml of 20% methanol followed by 1 ml of HPLC grade water/Milli-Q water. Elution of the analyte was performed with 1.0 ml of methanol. Evaporate the samples to complete dryness at a temperature below 50°C under gentle stream of nitrogen gas. Add 200 μL of sodium carbonate solution (40 mM) to the dried samples and vortex it for 2 min using Vibramax or vortexer. Add 200 μl dansyl chloride solution (1 mg/ml) and vortex it for 2 min using Vibramax or vortexer. The sample was kept for incubation for 15 min at approximately 60°C in nitrogen evaporator/water bath/incubator. The sample was allowed to attain room temperature and further subjected to liquid–liquid extraction (LLE) procedure.

Liquid–liquid extraction

Add 2.5 ml of methyl-tert-butyl ether (TBME) and shake for 10 min on vibromax at 2500 rpm/Multi-Pulse vortexer (5 min with pulse and 5 min without pulse) at 80 rpm with medium frequency. Then, centrifuge the samples at 3000 rpm and at 10°C for 5 min. Transfer the organic layer into prelabeled glass tubes and evaporate the samples to complete dryness under a stream of nitrogen gas at ≤50°C. The residue was reconstituted in 300 μl of reconstitution solution. The samples were transferred into HPLC vials for analysis and 10 μl was injected on to LC/MS/MS system.

Tandem Mass Spectrometry (MS/MS) conditions

For operation in MS/MS mode, a mass spectrometer fitted with an electrospray ion source interface was used for analysis. The source was operated initially at a temperature of 400°C. The mass spectrometer was programmed to monitor the protonated molecule [M + H]+ at m/z 530.30 via the first quadrupole filter (Q1), the product ion at m/z 171.10 was monitored via the third quadrupole filter (Q3). Finally, all MS parameters were manually fine tuned to obtain the highest MRM signals. The MRM transition m/z 530.30/171.10 was monitored for the detection of Ethinyl Estradiol. Internal standard also processed in the same way as that of Ethinyl Estradiol. 500 ng/ml solution of Analyte and Internal standard solution were separately used for tuning. And these solutions are injected into mass spectrometer directly, and the mass spectrometer was operated in the positive mode with the following parameters. Manual tuning was done for both Ethinyl estradiol and internal standard by using optimized conditions mentioned in [Table 1] for MS detector.
Table 1: Optimized MS conditions

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Optimized chromatographic conditions

The determination of EE in human plasma was carried out using SB C18 HT (50 mm × 3.0 mm) 1.8 μm column as stationary phase, and a composition of 2 mM ammonium formate buffer: acetonitrile (20:80 v/v) was used as mobile phase with flow rate of 0.3 ml/min. 10 μl sample volume was injected into system for each run. The total run time was 4.0 min. The column temperature was maintained at 60°C.

Data collection and integration

All integrations were performed by Applied Biosystems Analyst Software Version 1.5. The slopes, intercepts, and correlation coefficients were determined by least squares linear regression analysis using the ratios of drug/ISTD peak areas of calibration curve standards. A weighting factor of was used in the calculation of the linear regression line. All the QC samples were also calculated by Applied Biosystems Analyst Software Version 1.5. (Thermo Fisher Scientific). To prepare the tables, the concentrations were exported directly to the Microsoft Excel, and from the excel sheet, the data are transferred into individual tables with 100% data check. The report was prepared using Microsoft Word and all the entries were verified against the raw data.


  Method Validation Top


The method was validated for selectivity, linearity, accuracy, precision, recovery, and stability according to the International Conference on Harmonization guidelines.[21],[22] Validation runs were conducted on 3 consecutive days. Each validation run consisted of one set of CSs and six replicates of QC plasma samples. The selectivity of the method was evaluated by analyzing six blank Human plasma, blank plasma-spiked EE and IS, and a human plasma sample.

Calibration curves were constructed by analyzing spiked calibration samples on 3 separate days. Peak area ratios of EE to IS (EE-d4) were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 5–308.560 pg/ml for EE. The LLOQ was defined as the lowest concentration on the calibration curves, which can be quantified reliably, with an acceptable accuracy (80%–120%) and precision (<20%).

Accuracy and precision were assessed by the determination of QC samples at five concentration levels in six replicates (5.20, 15.60, 44.88, 89.76, and 179.52 pg/ml) on 3 validation days. The precision was expressed by the coefficient of variation (%CV).

The recovery of EE was evaluated by comparing peak area ratios of QC samples with those of reference QC solutions reconstituted in blank plasma after extraction (n = 6). The recovery of the IS was determined in a similar way.

To evaluate the matrix effect, blank human plasma after extraction and then spiked with the analyte at QC level was used. The corresponding peak areas were then compared with those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the matrix effect. The matrix effect of IS was evaluated at eight different concentrations in the same manner. Matrix sources were from six lots of blank human plasma.

Carry over was assessed following injection of a blank plasma sample immediately after three repeats of the upper limit of quantification, and the response was checked.

The stability of Ethinyl Estradiol in human plasma was evaluated by analyzing three replicates of plasma samples at the concentrations of 15.60 and 179.52 pg/ml which were exposed to different conditions. These results were compared with those obtained for freshly prepared plasma samples. The bench top stability was determined after exposer to room temperature for about 24 hr, 25 min. The coolant stability was determined by placing samples in dry ice for approximately about 45 hr 15 min, Post extracted refrigerator stability was determined by placing samples in matrix at 2-80° C, short-term stability was determined after the exposure of the spiked samples at room temperature for about 10 h, 30 min and the ready-to-inject samples (after extraction) stability was determined by placing samples in the HPLC auto sampler at 5° C for 9 h 52 min. The freeze–thaw stability was evaluated after four complete freeze–thaw cycles (-70 ± 15°C & -20 ± 5°C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at 2-8°C for 26 days.


  Results and Discussion Top


Optimization of LC-MS/MS conditions

In the present study, the LC-MS/MS analysis was performed in positive ion MRM mode. The positive ion MRM mode provided better signal-to-noise ratios and was more suitable for quantitative determination. Parameters such as collision energy, desolvation temperature, electrospray ionization source temperature, capillary and cone voltage, flow rate of desolvation gas, and cone gas were optimized to obtain the highest intensity of molecular ions of analytes. The product ion scan spectra of [MH] for EE showed that the most abundant parent ion m/z and product ion m/z were 530.13 and 171.10, respectively. However, the product ion scan spectra of [MH] for EE-d4 showed that the most abundant parent ion m/z and product ion m/z were 534.11 and 171.15, respectively. The chromatograms of the human plasma, analyte, and ISTDs were shown in [Figure 2].
Figure 2: Representative chromatograms of (a) Human plasma, (b) Analyte, and (c) Internal standards

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The use of small particles of stationary phase below 5 μm allowed LC to push the limits of both peak capacity and speed of analysis without compromising resolution. Various mobile-phase conditions were evaluated to obtain optimized responses, suitable retention times, good peak shapes, and satisfying resolutions for the analytes.

It was observed that the accuracy is accepted for calibration curve standards, extraction recovery was good, so TBME was selected as an extracting solvent. However, analyte interference was found at blank.

Sample extraction

SPE and LLE were used for sample preparation. Although various SPE cartridges, solvents, and mixture of solvents were tested, it was observed that the interference in hydrophilic lipophilic balanced (HLB), mixed-mode polymeric sorbent (MCX) cartridges was less, but still interference was found in blank. 0.2% formic acid was used as extraction additive in SPE. The result obtained was not satisfactory in HLB cartridges when compared to result found better in MCX cartridges as no interference was found at the analyte Rt and ISTD Rt. As the result was good with MCX cartridges, MCX cartridges were selected in the first step of SPE followed by TBME as an extraction reagent in LLE.


  Method Validation Top


Selectivity and linearity

[Figure 2] shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with EE, and IS. No interference of endogenous substance was observed at the retention time of the EE and IS, the results were shown in [Table 2] and [Table 3].
Table 2: Summary of calibration curve parameters for ethinyl estradiol (analyte) in human plasma

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Table 3: Back-Calculated standard concentrations from each calibration curve for ethinyl estradiol in human plasma and in the presence of ethinyl estradiol-D4

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The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range of 5–308 pg/ml for EE in human plasma. A typical equation of the calibration curve was: y = 0.0214x + 0.0466, r2 = 0.9981 for EE, where y represents the ratios of analyte peak area to that of IS and x represents the plasma concentration. [Figure 3] and [Figure 4] show the linearity curve and typical chromatograms of linearity, respectively. The LLOQ for EE in human plasma was 16 pg/ml. The LLOQ for EE in human plasma was found to be 5.2 pg/ml.
Figure 3: Calibration curve of ethinyl estradiol

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Figure 4: Representative chromatograms of calibration curve

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Precision

The precision of the method was determined by calculating CV for QCs at three concentration levels over 3 validation days. Intra-day precision was 17.73% or less and the inter-day precision was 10.86% or less at each QC level, the results were shown in [Table 4]. The accuracy of the method ranged from 86.91% to 105.56% at each QC level. The results were shown in [Table 4].
Table 4: Precision for determination of ethinyl estradiol levels in human plasma

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Recovery and matrix effect

Mean recoveries of EE at concentrations of 179.520, 89.760, and 44.880 pg/ml were measured to be 70.02% ± 7.81%, 70.86% ± 3.81%, and 76.42% ± 8.04% (n = 6), respectively. The results were depicted in [Table 5]. The recovery of the IS (200 ng/ml) was measured to be 66.30% ± 7.80%. The results were depicted in [Table 5].
Table 5: Recovery of analyte (ethinyl estradiol) and internal standard (ethinyl estradiol-d4) from female human K2 EDTA plasma

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The matrix effects for EE at concentrations of 15.600, 44.880, 89.760, and 179.520 were measured to be 100.50 ± 2.6, 88.57 ± 3.08, 88.14 ± 2.17, and 89.89 ± 1.67, respectively. The matrix effect for EE-d4 (IS) was measured to be 92.34 ± 13.70. The matrix effect of human plasma was found negligible on analyte recovery.

Ion suppression and enhancement of analytes and internal standard

The %CV for ion suppression and enhancing of EE for unextracted and postextracted is 6.91% and 7.72, respectively. The %CV for ion suppression and enhancing of IS for unextracted and postextracted is 6.65% and 12.39%, respectively. The percentage ion suppression and enhancing of EE and ISTD is 10.24% and 10.43%, respectively.

Dilution integrity

The %accuracy of EE nominal concentrations was 101.13% and 102.98% for 1 in 2 dilutions and 1 in 4 dilutions, respectively. The %CV was 0.77%–2.25%.

Partial volume analysis

The accuracy for 1/2th and 1/4th concentration of EE HQC was 100.28% and 104.46%, respectively. The precision (%CV) for 1/2th and 1/4th concentration of EE HQC was 4.29% and 2.31%, respectively.

Whole batch re-injection reproducibility

The accuracy of EE QC samples in reinjection was from 91.80% to 101.20%. The precision (%CV) of EE QC samples in reinjection was from 1.40% to 6.44%. EE was found to be stable at room temperature postextraction (in reconstitution solution) for approximately 15 h, 52 min and reproducible after reinjection.

Injector carryover effect for analyte and internal standard

No significant injector carryover is observed for EE and ISTD.

Ruggedness-different analyst

The accuracy of EE QC samples was within the range of 93.83%–113.00%. The precision of EE QC samples was within the range of 1.59%–9.63%. These results indicated that the method is rugged and reproducible by different analysts and different columns.

Stability

The QC samples of EE were subjected to various stability studies such as benchtop stability (at room temperature for 24 h, 25 min), stability of analyte in biological matric in dry ice (coolant stability), postextracted refrigerator stability in matrix at 2°C–8°C, dry extract stability, freeze and thaw stability (after 4th cycle at −70°C ± 15°C and −20°C ± 5°C), in-injector stability at 5°C in autosampler for approximately 9 h, 52 min, and stock solution stability (short- and long-term). The results indicated that the EE was stable under the above-mentioned conditions since the percentage assay changes were within ±5%. Results were shown in [Table 6].
Table 6: Assessment of stability of analyte (ethinyl estradiol) at different conditions

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  Conclusions Top


High-sensitive LC-MS/MS method for the analysis of oral contraceptive EE was developed and validated over the curve range of 5.0–310 pg/ml using 400 μl of human plasma sample. EE and the ISTD, EE-d4, were extracted from the plasma matrix with efficient SPE, derivatized with dansyl chloride, and then back extracted into TBME. The rugged, efficient SPE method followed by LLE provides exceptional sample cleanup and high recoveries with a runtime of 4 min only. The high extraction efficiency, low limit of quantization, and wide linear dynamic range make this a suitable method for use in clinical samples from bioequivalence studies following oral administration of EE during bioequivalence studies on EE tablets in healthy human subjects. This high sensitivity method can also be used for therapeutic drug monitoring or supporting bioequivalence and drug–drug interaction studies in human subjects.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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