Ethinyl Estradiol and Drospirenone in Plasma. AB SCIEX Triple Quad⢠5500 System and UPLC Chromatography. Siddharth Khurana1, Naveen Dubey1, ...
Highly Sensitive and Robust Quantification Method for Ethinyl Estradiol and Drospirenone in Plasma AB SCIEX Triple Quad™ 5500 System and UPLC Chromatography Siddharth Khurana1, Naveen Dubey1, Dhananjay Sharma1, Sandeep Sharma1, Pravin Khadke2, Anoop Kumar2 and Manoj Pillai2 1 Jubilant Clinsys Ltd, C-46, Sector 62, Noida, UP, India 2 AB SCIEX, 121, Udyog Vihar, Phase IV, Gurgaon, Haryana, India
Introduction Estrogens and progesterone play an important role in fertility and sexual development, as well as in cancer risk. In modern world estrogen and progesterone combination based drugs are widely used as most effective contraceptive agents. One of such combination is Ethinyl estradiol (EE, Figure 2a) and Drospirenone. The mean bioavailability of EE is reported to be 45%. EE is known to undergo extensive metabolism and also can be highly protein bound makes its bio availability very low in human system. Drospirenone (Figure 2b) is a novel synthetic progestogen with a pharmacological profile similar to its natural progesterone. It will be metabolized completely but the metabolites are not shown any biological activities and undergo hepatic and renal elimination. Drospirenone and ethinyl estradiol combination in formulation have similar efficiency and safety profile to other low dose oral contraceptive. It has been also reported that it has less side effect with regards to weight gain, mood change etc. Highly sensitive and accurate low level quantitation becomes essential for the bioequivalence studies for such molecules. The main objective of this work is to develop and validate a highly sensitive and reproducible method for EE TM (1.0 pg/ml) in human plasma using AB SCIEX Triple Quad 5500 LC/MS/MS system (Fig 1).
Key Features of the Method using AB SCIEX Triple Quad™ 5500 System •
A sensitive, reproducible and cost effective LC/MS/MS method was developed for a GLP bioanalytical laboratory using simple liquid-liquid extraction sample preparation method
•
The LLOQ for Ethinyl Estradiol in plasma was 0.993 pg/ml (10 fg on column) S/N ratio of 68 with good precision and accuracy for n = 12 inj. in human plasma
•
Accuracy and precision for Ethinyl Estradiol and Drospirenone are between 80-120% meeting the validation requirements for regulated bio analytical labs
•
New, patented QJet 2 Ion Guide improves sensitivity ® and robustness: The patented QJet 2 Ion Guide design yields improved ion containment and operates at higher pressures providing better collisional focusing to enhance ion transmission for ultimate sensitivity
•
Next-generation eQ™ Electronics on AB SCIEX Triple TM Quad 5500 provide improved performance at ultra-low MRM dwell times for improved support of fast LC and narrow peak widths.
®
O H3C O
HH HO
CH3
CH H
H
CH OH
H
H
3
O
H H H
H Figure 1: AB SCIEX Triple QuadTM 5500 system
Figure 2a: Ethinyl Estradiol
Figure 2b: Drospirenone
p1
acetonitrile:methanol (mobile phase B), at a flow rate of
Material and Methods
300μL/min. The total run-time for the method was 8.0 minutes.
Sample Preparation
The injection volume was set to 10μL. The rinsing solution was Plasma (500µL) samples were spiked (2%) with Ethinyl Estradiol
Methanol: Water (50/50) mixture.
(EE) and Drospirenone standard with 10µL internal standard solution. The samples were vortex with 2ml of TBME: n-Hexane 0
Mass Spectrometry conditions
mixture and centrifuged at 4 C. Organic phase were collected
An AB SCIEX Triple Quad™ 5500 LC/MS/MS system equipped
and evaporated to dryness under nitrogen stream. The residue in
with Turbo V
each tube were dissolved in the 200ul of Ammonium bicarbonate
Ionization (ESI) mode. The optimized MRM for the analyte and
(pH:11) followed by 2min vortex. Dansyl chloride (0.5 mg/mL
internal standard are summarized in Table 1. The source
solution in acetone) was added to derivatized the Ethinyl
parameters were optimized in flow injection analysis. Quadrupole
TM
source was used, in positive Electrospray
0
Estradiol. Reaction mixture tubes were kept for 10 min at 60 C in
mass analyzers (Q1 and Q3) were set at unit resolution for
water bath. LLE was again performed with TBME : n-Hexane
quantitative analysis. The mass spectrometer was operated in
(2mL) followed by vortex and centrifugation. Organic phase were
positive ionization mode with electrospray voltage +5500 V and
collected and evaporated and finally reconstituted in acetonitrile
source temperature of 550 C. Nitrogen was used as nebulizing
and water (200µL) for quantitation in AB SCIEX Triple Quad
TM
5500 LC/MS/MS system
o
gas (GS1), drying gas (GS2) and curtain gas at 50, 55 and 30 arbitrary unit, respectively. Valco valve was used to divert the flow to avoid the interference during the chromatographic run.
Results Ethinyl estradiol was derivatised with dansyl chloride to obtain the maximum sensitivity in plasma sample (Fig 3). Dansyl - EE produced the Q1MS ion m/z 530.2 and major product ion 171.1 which correspond to 5-(dimethylamino)-naphthalene moiety. The best ionization was achieved in ESI positive mode. The mass spectrometric parameters for both the compounds are given in
Figure 3: Chemical derivatization of Ethinyl estradiol using dansyl chloride
Table 1. A linear calibration curve was constructed using the 2
1/X
regression. The calibration curve for EE was linear over a
dynamic range of 0.993-300.48 pg/mL in plasma sample (Fig 8)
HPLC condition
with an r value 0.9980 and similarly, calibration curve for A Shimadzu Nexera with 30AC auto sampler system was used,
drospirenone shown in figure 9 has r value 0.9994
with a C18 (50 x 2.1 mm, 5μm) analytical column maintained at 30ºC. A gradient elution was employed, consisting of 5mM Ammonium Formate buffer (mobile phase A) and Table 1: MRM transitions and optimised MS parameter for compounds Analyte
MRM
Dwell Time (ms)
DP
EP
CE
CXP
Dansyl-Ethinyl Estradiol
530.2/171.1
200
100
15
49
16
D4 - Dansyl-Ethinyl Estradiol
534.2/171.0
200
100
15
49
16
Drospirenone
367.1/97.00
200
100
121
29
14
D4-Drospirenone
371.1/97.00
200
100
121
29
14
p2
Several precision and accuracy batches were processed to evaluate the developed method. Results of EE and Drospirenone for precision and accuracy in batch-1 were given in Table 3 and 4. Different QCs level samples (n = 12) were also evaluated for precision and accuracy along with the calibration curve. Results were shown in table 2. The EE and Drospirenone eluted at 0.88 min and 4.10 min with minimum background noise in an 8 min
No peak was observed at the RT of the analyte in extracted
chromatographic run time (Fig 4 and Fig 6). The signal to noise ratio of EE at LOQ (0.993 pg/mL) in plasma is 68.3 calculated using sigma standard deviation of the baseline (Fig 7).
Figure 6: The chromatograms for Dansyl-Ethinyl Estradiol in zero blank plasma sample (upper pane) and 0.993 pg/mL spiked in plasma sample (lower pane)
Figure 4: The chromatograms for drospirenone in zero blank plasma sample (upper pane) and 1003.63 pg/mL spiked in plasma sample (lower pane)
Figure 7: Signal to noise calculation for Dansyl-Ethinyl Estradiol in extracted plasma sample at LLOQ level (0.993 pg/mL) is 68.3
Figure 5: Signal to noise calculation for Drospirenone in extracted plasma sample at LLOQ level (1003.63 pg/mL) is 284 p3
Drospirenone
Ethinyl Estradiol
Analyte
Sample Name
Exp. Con (pg/ml)
LLOQ
0.9980
LQC -1 MQC -1 HQC -1 LLOQ LQC -1 MQC -1 HQC -1
Cal. Con (pg/ml)
Number of values used
%CV
Accuracy
1.0406
12
4.5702
104.27
2.6740
2.7786
12
1.6824
103.91
150.2430
149.2491
12
1.0126
99.33
246.3980
242.2303
12
2.2463
98.30
1004.0070
1052.0814
12
2.0557
104.78
2706.2190
2885.9109
12
0.8237
106.63
84569.3450
84438.5864
12
1.0554
99.84
140948.9090
141724.8942
12
2.464
100.55
Table 2: Statistic calculation of quality controls (n=12 inj) in human plasma sample for Dansyl-Ethinyl Estradiol and Drospirenone
Figure 8: Calibration curve of Dansyl-Ethinyl Estradiol in plasma from 0.993 pg/ml to 300.485 pg/ml . The method has shown excellent linerarity over the concentration range with r = 0.9980
Figure 9: Calibration curve of drospirenone in plasma from 1003.63 pg/ml to 171152.24 pg/ml . The method has shown excellent linerarity over the concentration range with r = 0.9994 p4
Sample Name
Sample Type
Analyte Area
IS area
Area Ratio
Actual Con. (pg/ml)
Cal Con. (pg/ml)
Accuracy (%)
BLANK BLANK DUP ZERO BLANK ZERO BLANK DUP STD A STD B STD C STD D STD E STD F STD G STD H LLOQ QC -1 LQC -1 MQC -1 HQC -1 BLANK-QC-1 LLOQ QC -2 LQC -2 MQC -2 HQC -2 BLANK-QC-2 LLOQ QC -3 LQC -3 MQC -3 HQC -3 BLANK-QC-3 LLOQ QC -4 LQC -4 MQC -4 HQC -4 BLANK-QC-4 LLOQ QC -5 LQC -5 MQC -5 HQC -5 BLANK-QC-5 LLOQ QC -6 LQC -6 MQC -6 HQC -6 BLANK-QC-6 LLOQ QC -1 LQC -1 MQC -1 HQC -1 BLANK-QC-1 LLOQ QC -2 LQC -2 MQC -2 HQC -2 BLANK-QC-2 LLOQ QC -3 LQC -3 MQC -3 HQC -3 BLANK-QC-3 LLOQ QC -4 LQC -4 MQC -4 HQC -4 BLANK-QC-4 LLOQ QC -5 LQC -5 MQC -5 HQC -5 BLANK-QC-5 LLOQ QC -6 LQC -6 MQC -6 HQC -6 BLANK-QC-6
Blank Blank Double Blank Double Blank Standard Standard Standard Standard Standard Standard Standard Standard QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank
0 0 0 0 6774 15652 285956 571083 1042239 1416537 1695288 2294041 7910 18848 1138887 1810539 0 8023 20786 1145245 1842821 0 7561 21328 1138909 1776442 0 7327 20886 1126520 1852672 0 7443 21344 1181971 1892347 0 7392 22130 1174469 1859869 0 8202 18658 1152367 1814875 0 7819 20760 1174976 1804859 0 8451 22429 1140860 1796568 0 7689 20964 1111755 1842642 0 7514 21109 1136565 1853768 0 7335 20904 1140076 1815216 0
0 0 938116 947065 1042864 1032269 1095472 1139339 1103685 1102616 1022132 1114136 1067614 1013308 1149211 1135065 0 1091544 1107128 1153842 1155252 0 1058303 1132382 1146045 1117713 0 1048775 1105252 1102080 1124222 0 1067437 1122133 1162917 1152492 0 1060293 1148796 1158475 1109429 0 1103094 1004330 1150717 1124453 0 1156027 1104472 1167045 1123271 0 1057894 1138019 1130733 1126934 0 1046702 1121431 1118328 1094014 0 1059129 1119136 1136572 1145757 0 1032385 1113784 1135968 1078222 0
0 0 0 0 0.0065 0.0152 0.261 0.5012 0.9443 1.2847 1.6586 2.059 0.0074 0.0186 0.991 1.5951 0 0.0074 0.0188 0.9925 1.5952 0 0.0071 0.0188 0.9938 1.5894 0 0.007 0.0189 1.0222 1.648 0 0.007 0.019 1.0164 1.642 0 0.007 0.0193 1.0138 1.6764 0 0.0074 0.0186 1.0014 1.614 0 0.0068 0.0188 1.0068 1.6068 0 0.008 0.0197 1.009 1.5942 0 0.0073 0.0187 0.9941 1.6843 0 0.0071 0.0189 1.0000 1.6179 0 0.0071 0.0188 1.0036 1.6835 0
0 0 0 0 0.993 1.987 37.486 74.971 150.243 193.646 253.743 300.485 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0 0.998 2.674 150.243 246.398 0
0 0 0 0 0.934 2.223 38.791 74.516 140.416 191.04 246.645 306.204 1.07 2.734 147.36 237.204 0 1.061 2.76 147.588 237.214 0 1.03 2.769 147.77 236.35 0 1.007 2.778 151.994 245.066 0 1.005 2.797 151.133 244.174 0 1.005 2.833 150.749 249.299 0 1.074 2.731 148.909 240.016 0 0.974 2.763 149.707 238.943 0 1.156 2.899 150.028 237.072 0 1.06 2.748 147.822 250.47 0 1.023 2.773 148.695 240.601 0 1.024 2.759 149.234 250.356 0
0 0 0 0 94.04 111.86 103.48 99.39 93.46 98.65 97.2 101.9 107.18 102.25 98.08 96.27 0 106.3 103.22 98.23 96.27 0 103.23 103.55 98.35 95.92 0 100.88 103.9 101.17 99.46 0 100.68 104.59 100.59 99.1 0 100.66 105.94 100.34 101.18 0 107.57 102.12 99.11 97.41 0 97.57 103.34 99.64 96.97 0 115.81 108.41 99.86 96.21 0 106.24 102.77 98.39 101.65 0 102.49 103.7 98.97 97.65 0 102.64 103.18 99.33 101.61 0
Table 3: Full precision and accuracy batch data for Dansyl-Ethinyl Estradiol in plasma sample
p5
Sample Name
Sample Type
Analyte Area
IS area
Area Ratio
Actual Con. (pg/ml)
Cal Con. (pg/ml)
Accuracy (%)
BLANK BLANK DUP ZERO BLANK ZERO BLANK DUP STD A STD B STD C STD D STD E STD F STD G STD H LLOQ QC -1 LQC -1 MQC -1 HQC -1 BLANK-QC-1 LLOQ QC -2 LQC -2 MQC -2 HQC -2 BLANK-QC-2 LLOQ QC -3 LQC -3 MQC -3 HQC -3 BLANK-QC-3 LLOQ QC -4 LQC -4 MQC -4 HQC -4 BLANK-QC-4 LLOQ QC -5 LQC -5 MQC -5 HQC -5 BLANK-QC-5 LLOQ QC -6 LQC -6 MQC -6 HQC -6 BLANK-QC-6 LLOQ QC -1 LQC -1 MQC -1 HQC -1 BLANK-QC-1 LLOQ QC -2 LQC -2 MQC -2 HQC -2 BLANK-QC-2 LLOQ QC -3 LQC -3 MQC -3 HQC -3 BLANK-QC-3 LLOQ QC -4 LQC -4 MQC -4 HQC -4 BLANK-QC-4 LLOQ QC -5 LQC -5 MQC -5 HQC -5 BLANK-QC-5 LLOQ QC -6 LQC -6 MQC -6 HQC -6 BLANK-QC-6
Blank Blank Double Blank Double Blank Standard Standard Standard Standard Standard Standard Standard Standard QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank QC QC QC QC Blank
0 0 0 0 43512 86613 1269594 2163306 4137593 5050928 6298565 8471100 46634 114721 3750300 6465514 0 46055 136812 3883127 6441550 0 44989 127136 3731672 6061461 0 43388 129349 3738489 6596685 0 46608 128794 4033150 6493276 0 46368 134761 4051400 6698465 0 44202 113168 3810063 6387153 0 46977 133094 3864471 6271133 0 45425 128284 3713134 6060603 0 41655 128933 3731948 6316049 0 46875 124722 3863058 6227108 0 43791 131589 3841234 6548602 0
0 0 723630 744557 827716 852938 871988 924851 909475 912364 808586 889543 820920 765308 837571 878482 0 850857 897367 860608 874753 0 820538 844085 836690 807118 0 800670 865128 853970 853086 0 864451 845264 897485 879457 0 847928 892569 894376 872771 0 831790 746900 849549 853936 0 871076 887061 856809 862339 0 816638 845304 827464 823800 0 794816 843980 845906 814215 0 859705 828209 875682 851013 0 818559 857424 865575 844062 0
0 0 0 0 0.0526 0.1015 1.456 2.3391 4.5494 5.5361 7.7896 9.523 0.0568 0.1499 4.4776 7.3599 0 0.0541 0.1525 4.5121 7.3638 0 0.0548 0.1506 4.46 7.51 0 0.0542 0.1495 4.3778 7.7327 0 0.0539 0.1524 4.4938 7.3833 0 0.0547 0.151 4.5299 7.6749 0 0.0531 0.1515 4.4848 7.4797 0 0.0539 0.15 4.5103 7.2722 0 0.0556 0.1518 4.4874 7.3569 0 0.0524 0.1528 4.4118 7.7572 0 0.0545 0.1506 4.4115 7.3173 0 0.0535 0.1535 4.4378 7.7584 0
0 0 0 0 1003.632 2007.263 27686.393 43794.84 85576.123 110745.571 145982.798 171152.246 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0 1004.007 2706.219 84569.345 140948.909 0
0 0 0 0 1019.248 1944.923 27544.06 44235.05 86011.23 104659.3 147251.4 180012.7 1099.332 2858.855 84653.37 139129.4 0 1048.701 2907.201 85305.11 139204.5 0 1061.958 2872.442 84321.71 141967 0 1049.878 2851.54 82766.89 146176.4 0 1044.697 2905.542 84960.43 139571.7 0 1059.213 2879.25 85641.31 145084.3 0 1030.06 2889.395 84789.83 141393.4 0 1044.958 2861.451 85271.69 137473 0 1076.98 2893.994 84838.17 139072.9 0 1016.203 2913.026 83409.52 146639.4 0 1056.212 2871.923 83404 138324.4 0 1036.786 2926.313 83901.02 146662.4 0
0 0 0 0 101.56 96.89 99.49 101.01 100.51 94.5 100.87 105.18 109.49 105.64 100.1 98.71 0 104.45 107.43 100.87 98.76 0 105.77 106.14 99.71 100.72 0 104.57 105.37 97.87 103.71 0 104.05 107.37 100.46 99.02 0 105.5 106.39 101.27 102.93 0 102.59 106.77 100.26 100.32 0 104.08 105.74 100.83 97.53 0 107.27 106.94 100.32 98.67 0 101.21 107.64 98.63 104.04 0 105.2 106.12 98.62 98.14 0 103.26 108.13 99.21 104.05 0
Table 4: Full precision and accuracy batch data for Drospirenone in plasma sample p6
Conclusions
•
A highly sensitive method for Ethinyl Estradiol and Drospirenone was developed and validated using AB TM SCIEX Triple Quad 5500 LC/MS/MS system in human plasma. The developed method is sensitive, reproducible and cost effective for bioanalytical laboratory with good precision and accuracy
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