Keywords

LC-MS/MS method development, Solid phase extraction procedure, Hypurity C18 column

Introduction

Mifepristone (RU38486) is a progesterone receptor antagonist clinically. It is a synthetic drug used as an abortifacient. It is a 19-norsteroid with potent competitive anti-progestational and significant anti-glucocorticoid as well as anti-androgenic activity. RU38486 used to reduce glucocorticoid receptor activation on potential therapy in the metabolic syndromes and depression, used as investigational tool to dissect hypothetical-pituitary axis which regulates glucocorticoid production. The dose used for anti-glucocorticoid therapy is comparatively less compared to gynecological purpose [1]. Previous method of quantitative analysis of RU38486 includes radio immune assay however antibody used has cross reactivity provides more preference for HPLC analysis. Homer et al. developed a quantitative analysis of RU38486 by HPLC triple quadrapole mass spectrometry with a linearity range of 0.5-500 ng (r2=0.997) and a Limit of Detection (LOD) at 50 pg injected on column [2]. Guo et al. developed an HPLC method for the determination of mifepristone in human plasma with a linearity range of 10 ng/ml to 20 μg/ml (r2=0.9991) and an LOD of 6 ng/ml [3] whereas Tang et al. was developed a method for simultaneous determination of mifepristone and monodemethyl mifepristone in human plasma by liquid chromatography-tandem mass spectrometry method using levonorgestrel as internal standard with a linearity range of 5-2000 ng/ml for mifepristone and monodemethyl mifepristone with an LOD of 1.0 ng/ml for both analytes [4]. Whereas Guo et al. developed a HPLC method for simultaneous determination of rivanol and mifepristone in human plasma by solid phase extraction method, linearity was found as 5-100000 ng/ml and an LOD of 3 ng/ml [5]. In this project aimed to develop a highly specific and accurate method for analysis of mifepristone and its metabolites(N-demethy mifepristone and hydroxyl mifepristone) in human plasma by using LC-MS/MS (Tables 1 and 2).

Table 1: Specificity and selectivity test for mifepristone analyte and internal standard (Aqueous)

Table 2: Specificity and selectivity test for mifepristone analyte and internal standard (Spiked)

Materials and Methods

Mifepristone was purchased from vivan life sciences, N-demethyl mifepristone (RU 42 633) and hydroxyl mifepristone (RU 42 698) was purchased from TLC pharma chem.., Inc. The internal standard levonorgestrel was purchased from Vivan life Sciences. HPLC grade methanol and phosphoric acid purchased from Fisher Scientific, HPLC grade acetic acid purchased from Merck. Plasma was purchased from Laxmi Sai Clinical lab, India and HPLC grade Milli Q water was produced in house by using Milli Q-RO named Millipore. Solid phase extraction cartridge (Oasis HPLC 1 ml 30 mg), the HPLC separation column was HyPURITY c18, 50 × 4.6 μm, 5 μm from thermo scientific, centrifuge of 5810R, micropipettes of variable adjustment from eppendrof, vortex mixer of vibramax 110 from Heidolph, Microbalance of MX5 from Mettler Toledo, Deep freezer of -75°C Deep freezer from Sanyo and Low volume evaporator of Turbovap^® (LV) from Caliper life sciences (Tables 3 and 4).

Table 3: Back calculated calibration curve concentrations for mifepristone and internal standard

Table 4: Back calculated calibration curve concentrations for N-demthyl Mifepristone and internal standard

Chromatographic system

The LC-MS/MS system 6460 of Triple Quard from Agilent with an automatic injecting system was used. System management and hardware interface for data management from agilent [6]. The mobile phase was combination of Methanol: Water containing 0.2% acetic acid (75:25 v/v) Column oven temperature was 25°C, Auto sampler temperature was 5°C, flow rate was set at 0.5 ml/min, and injection volume was set at 2 μl with a run time of 4 min.

Preparation of stock solutions and working solutions

Stock solution of Mifepristone was prepared in diluents combination of methanol: water (90:10 v/v) and stored in clean glass bottle (Figures 1 and 2). They were later stared at 5°C until they were used for the preparation of working solutions by appropriate volume of diluents. Short term stock solution stability was carried out for all analytes (RU 38 486, RU 42 633 and RU 42 698) after storing it at room temperature for 2 hrs against freshly prepared samples (Table 5) [7].

Table 5: Back calculated calibration curve concentrations for RU 42 698 and Internal standard

Figure 1: Representative chromatogram of aqueous samples of Mifepristone(A), N-demethyl mifepristone(B) and Hydroxy Mifepristone(C) respectively

Figure 2: Representative chromatogram of a blank matrix Sample with internal standard for Mifepristone, N-demethyl Mifepristone and Hydroxy Mifepristone respectively

Preparation of calibration standards and QC samples in plasma

Calibration standards was prepared at a concentration rage of 52.4332-4100.9056 ng/ml for mifepristone, 54.5042- 4262.8800 ng/ml for N-demithyl mifepristone and 13.1661-1029.7455 ng/ml for hydroxy mifepristone by spiking 0.2 ml of each and made to 10 ml by adding diluents (Methanol: Water): 50:50 v/v (Diluents), QC samples was prepared by adding 0.2 ml of each and made to 10 ml by adding diluents (Figures 3 and 4) [8].

Figure 3: Representative chromatogram of aqueous samples of Mifepristone(A), N-demethyl mifepristone(B) and Hydroxy Mifepristone(C) respectively

Figure 4: Representative chromatogram of aqueous samples of Mifepristone(A), N-demethyl mifepristone(B) and Hydroxy Mifepristone(C) respectively Representative chromatogram of STD 1 sample for Mifepristone(A), N-demethyl Mifepristone (B) and Hydroxy Mifepristone(C) respectively

Extraction procedure

ll samples, including blank, unknown and standard was extracted using Oasis HLB cartridge of 1 ml, 30 mg before analysis. The optimized extraction procedure was as follows. Set HLB cartridge, condition the cartridge with 1 ml of 100% methanol, equilibrate the cartridge with 1 ml of water (MilliQ water 100%), load the sample in to HLB cartridge (3 mg) 1CC Oasis cartridge, elute with elution solvent (100% methanol), evaporate the eluent to dryness at 40°C and 20 psi nitrogen using low volume evaporator, reconstitute the dried residue with 0.2 ml of mobile phase, transfer the sample into appropriately labeled auto sampler vials and load the samples in to LC-MS/MS system [9].

Results and Discussion

Specificity/Selectivity

A series of chromatograms were shown in Figure 4 of all analytes including the internal standard in auto scale mode, Figure 2 shows the blank chromatograms which indicate the lack of interference, the method were identified as specific for analytes. The retention times are 2.129, 1.577, and 1.615 for mifepristone, N-demethyl mifepristone, hydroxyl mifepristone respectively and 2.562 for internal standard (levonorgestrel). Figure 1 was for mifepristone, N-demethyl mifepristone and hydroxyl mifepristone at concentrations of 52.4334, 54.5042 and 13.1661 ng/ml respectively [10].

Standard curve and linearity of the method

Calibration standards in plasma was prepared for analytes in the ranges of 52.43-4100.90 ng/ml for mifepristone, 54.50- 4262.88 ng/ml for N-demethyl mifepristone and 13.16-1029.74 ng/ml for hydroxyl mifepristone and an equal concentration of levonorgestrel was spiked to all the analytes. From the above standard graph LLOQ, LQC, MQC and HQC were found respectively (53.80, 158.24, 1883.85 and 3588.29 ng/ml for mifepristone), (55.92, 164.49, 1958.26 and 3730.02 ng/ml for N-demethyl mifepristone), (13.58, 39.96, 475.74 and 906.17 ng/ml for hydroxyl mifepristone) [11].

Accuracy and precision

The accuracy and precision was carried out. Inter day accuracy and precision was carried out by four QC as LOQQC, LQC, MQC and HQC respectively by six replicates on each analyte in three days, results are listed in Tables 6-9, intraday precision and accuracy was carried out on the same QC’s listed and was run by gape as six replicates on each analyte. All the readings are within the limit according to the guidelines [12-15].

Table 6: Lower Limit of quantification for Mifepristone, N-demethyl mifepristone and Hydroxy Mifepristone respectively

Table 7: Inter batch precision and accuracy for Mifepristone

Table 8: Inter batch Precision and accuracy for N-demethyl Mifepristone

Table 9: Inter batch Precision and accuracy for Hydroxy Mifepristone

Conclusion

A rapid, specific/selective and accurate method by liquid chromatography ESI triple quard mass spectrometry for the analysis of mifepristone and its metabolites (N-demethyl mifepristone and hydroxyl mifepristone) was freshly developed and validated in human plasma [16-20]. The assay used levonorgestrel as internal standard. The method was found linear in the range of 51.89 ng/ml to 4059.14 ng/ml, 51.90 ng/ml, 4059.69 ng/ml, 12.68 ng/ml, 992.36 ng/ml for Mifepristone, N-demethyl Mifepristone, and Hydroxy Mifepristone respectively in Biological Matrix using Solid phase extraction procedure. The LLOQ was found to be 50.95, 54.11 and 12.81 ng/ml for Mifepristone, N-demethyl Mifepristone, and Hydroxy Mifepristone respectively. The run time was fixed at 4.5 with peaks of superior resolution for all the analytes. The result shows that this developed and validated method can be used for routine examination of large number of biological samples [19-22].

Acknowledgment

All the praise and gratitude to the almighty God. The research work was supported by Azidus laboratories Ltd Chennai, would like to have my sincere gratitude to them all. My sincere thanks to Dr. S. Jasemine, Mr. Anand Babu and Mrs. Jisha Mol who helped me a lot to the way through.

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