LC-MS/MS Method Development and Validation of Mifepristone and its Metabolites in Human Plasma

Muhammed Rafsal KK* and Sijo Pattam

National College of Pharmacy, Manassery, Kerala, India

*Corresponding Author:
Muhammed Rafsal KK
National College of Pharmacy
Manassery, Kerala, India
 
Visit for more related articles at Der Pharmacia Sinica

Abstract

A rapid, specific and sensitive liquid chromatography-ESI mass spectrometry method was developed and validated for determination mifepristone and its metabolites in human plasma using levonorgestrel as internal standard. The extraction was performed by solid phase extraction procedure using waters Oasis HLB cartridge (1 cc, 30 mg). The column used was Hypurity C18 column (50 × 4.6 mm, 5 µm), the mobile phase was a combination of methanol water containing 0.2% Acetic acid at a ratio of 75:25 in isocratic mode. The flow rate was 0.5 ml/min with an injection volume of 2 µl and the total run time was 4 min. The detection was performed on a triple guard electron spray ionization mass spectrometry by selective reaction monitoring (SRM) mode. The target ions were monitored at [m+H] + m/z 430.3→134, 416.3→120, 446.3→109.1 for Mifepristone, N-Demethyl Mifepristone and Hydroxy Mifepristone respectively. Linearity was established in the range of 51.89 ng/ml to 4059.14 ng/ml, 51.9063 ng/ml to 4059.69 ng/ml, 12.68 ng/ml to 992.36 ng/ml for the determination of Mifepristone, N-Demethyl Mifepristone and Hydroxy Mifepristone respectively in K3EDTA using solid Phase Extraction procedure. The lower limit of quantification was found and reproducible (LLOQ) at 50.95 ng/ml, 54.11 ng/ml and 12.81 ng/ml for Mifepristone, N-Demethyl Mifepristone and Hydroxy Mifepristone respectively.

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).

  Mifepristone N-demethyl Mifepristone Hydroxy Mifepristone
Analyte Area IS Area Analyte Area IS Area Analyte Area IS Area
  1275 16611 1485 16611 462 16611
  1270 16406 1461 16406 408 16406
- 1376 18081 1457 18081 505 18081
  1305 18284 1450 18284 439 18284
  1382 17615 1485 17615 445 17615
  1186 19392 1493 19392 426 19392
AVERAGE 1299.00 17731.50 1471.83 17731.50 447.50 17731.50
SD 73.54 1114.51 17.94 1114.51 33.52 1114.51
% CV 5.66 6.29 1.22 6.29 7.49 6.29

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

  Mifepristone N-demethyl Mifepristone Hydroxy Mifepristone
Analyte Area IS Area Analyte Area IS Area Analyte Area IS Area
  1048 14361 1177 14361 336 14361
  1197 13799 1146 13799 371 13799
- 1062 14197 1314 14197 375 14197
  1139 16134 1499 16134 398 16134
  1020 14515 1285 14515 388 14515
  1110 16288 1366 16288 351 16288
  1226 15454 1315 15454 350 15454
  1186 15014 1237 15014 349 15014
AVERAGE 1129.500 14970.2500 1292.3750 14970.2500 364.7500 14970.2500
SD 76.000 916.11661 111.45266 916.11661 21.61844 916.11661
% CV 6.72 6.12 8.62 6.12 5.93 6.12

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).

Standard ID Std-1 Std-2 Std-3 Std-4 Std-5 Std-6 Std-7 Std-8
Nominal Concentration (µg/ml) 51.8992 103.7984 259.496 576.6578 1441.6444 2110.7532 3247.3126 4059.1408
Back Calculated Concentration (µg/ml) PA-02 50.5810 112.2690 234.4243 600.9680 1433.4766 2177.8631 3150.5721 4066.9174
PA-03 53.6505 94.8508 278.3877 550.8086 1383.7387 2007.7490 3274.0338 4486.2422
PA-04 53.6027 96.8367 265.6633 543.1534 1467.6403 2108.0269 3153.1109 4387.3201
AVERAGE 52.6114 101.3188 259.4918 564.9767 1428.2852 2097.8797 3192.5723 4313.4932
SD 1.75856 9.53496 22.62214 31.40354 42.19103 85.50980 70.55918 219.19421
%CV 3.34 9.41 8.72 5.56 2.95 4.08 2.21 5.08
% ACCURACY 101.37 97.61 100.00 97.97 99.07 99.39 98.31 106.27

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

Standard ID Std-1 Std-2 Std-3 Std-4 Std-5 Std-6 Std-7 Std-8
Nominal Concentration (µg/ml) 51.9063 103.8126 259.5315 576.7367 1441.8416 2111.0419 3247.7568 4059.696
Back Calculated Concentration (µg/ml) PA-02 54.8809 93.3984 243.9745 592.6648 1534.5458 2088.3308 3273.4116 4116.1187
PA-03 53.8270 94.8954 270.2119 572.2216 1366.9640 1999.7207 3295.9596 4487.5563
PA-04 53.4805 100.3317 258.7929 463.9041 1552.9200 2158.4919 3229.4398 4497.3802
AVERAGE 54.0628 96.2085 257.6598 542.9301 1484.8099 2082.1811 3266.2703 4367.0184
SD 0.72938 3.64843 13.15535 69.19768 102.47022 79.56406 33.83000 217.34106
%CV 1.35 3.79 5.11 12.75 6.90 3.82 1.04 4.98
% ACCURACY 104.15 92.68 99.28 94.14 102.98 98.63 100.57 107.57

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].

Standard ID Std-1 Std-2 Std-3 Std-4 Std-5 Std-6 Std-7 Std-8
Nominal Concentration (µg/ml) 12.6881 25.3762 63.4405 140.9789 352.4473 516.0282 793.8896 992.362
Back Calculated Concentration (µg/ml) PA-02 12.8824 24.1474 64.8572 148.5087 356.0282 518.0672 766.9526 969.7274
PA-03 13.1046 23.1908 67.2153 140.8820 340.6015 483.3853 791.6380 1085.8252
PA-04 12.6910 25.4607 66.4779 118.7894 374.9019 515.7603 762.2273 1074.3844
AVERAGE 12.8926 24.2663 66.1835 136.0601 357.1772 505.7376 773.6060 1043.3124
SD 0.20703 1.13962 1.20630 15.43530 17.17906 19.39200 15.79392 63.98265
%CV 1.61 4.70 1.82 11.34 4.81 3.83 2.04 6.13
% ACCURACY 101.61 95.63 104.32 96.51 101.34 98.01 97.45 105.13

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

der-pharmacia-sinica-aqueous-samples

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

der-pharmacia-sinica-matrix-Sample

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].

der-pharmacia-sinica-chromatogram-aqueous

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

der-pharmacia-sinica-demethyl-mifepristone

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].

Standard ID Std-1 S/N Ratio Standard ID Std-1 S/N Ratio Standard ID Std-1 S/N Ratio
Actual Concentration (µg/ml) 51.8992 - Actual Concentration (µg/ml) 51.9063 - Actual Concentration (µg/ml): 12.6881 -
PA-02 50.581 0.78 PA-02 54.8809 0.78 PA-02 12.8824 0.78
PA-03 53.6505 1.4 PA-03 53.827 1.4 PA-03 13.1046 1.4
PA-04 53.6027 1.41 PA-04 53.4805 1.41 PA-04 12.691 1.41
Stability 52.6163 10.4 FTS_IIS 53.5262 10.4 FTS_IIS 12.3308 10.4
PA-O1_RIR 44.3003 0.66 PA-O1_RIR 54.8355 0.66 PA-O1_RIR 13.0487 0.66
Average 50.9501 - Average 54.11 - Average 12.8115 -
SD 3.91985 SD 0.69603 SD 0.31338
%CV 7.69 %CV 1.29 %CV 2.45
% ACCURACY 98.17 % ACCURACY 104.25 % ACCURACY 100.97

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

QC ID LOQQC LQC MQC HQC
Nominal Concentration (µg/ml) 52.0203 153.0010 1758.6322 3349.7756
Back
Calculated Concentration    (µg/ml)
P & A 01 58.79 146.69 1642.93 2978.84
58.66 150.57 1582.76 3085.04
49.64 152.44 1641.57 2973.25
56.44 149.38 1627.77 3092.29
51.64 131.47 1623.95 3131.48
49.16 144.50 1629.22 3176.94

Table 7: Inter batch precision and accuracy for Mifepristone

QC ID LOQQC LQC MQC HQC
Nominal Concentration (µg/ml) 53.6093 157.6745 1812.3505 3452.096
Back
Calculated
Concentration 
(µg/ml)
P & A 02 59.19 160.09 1695.09 3164.67
53.10 148.54 1628.07 3304.69
55.40 160.39 1695.82 3028.08
62.61 134.20 1748.57 3283.26
56.96 152.17 1658.95 3268.16
56.88 146.05 1689.99 3329.34

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

QC ID LOQQC LQC MQC HQC
Nominal Concentration (µg/ml) 13.0977 38.5228 442.7906 843.4106
Back
Calculated
Concentration
(µg/ml)
P & A 02 13.1178 39.4054 389.8331 735.2812
13.1564 39.9848 369.5023 736.2313
12.9913 37.3303 388.4016 744.1352
12.7259 34.1807 391.2912 719.5374
12.4704 34.6297 380.4300 753.0830
12.6959 33.4218 391.0660 735.2019

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.

References

Select your language of interest to view the total content in your interested language

Viewing options

Flyer image

Share This Article