462 Original Article
A Simple, Rapid and Sensitive UFLC-MS/MS Method for the Quantification of Oral Contraceptive Norgestrel in Human Plasma and its Pharmacokinetic Applications
Affiliations
Key words ▶ norgestrel ● ▶ oral contraceptive ● ▶ UFLC-MS/MS ● ▶ method validation ● ▶ pharmacokinetics ●
received 29.10.2013 accepted 15.11.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1361164 Published online: December 5, 2013 Drug Res 2014; 64: 462–469 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence L. M. K. Pallapothu Deputy Director, Bioanalytical Aizant Drug Research Solutions (P) Ltd. Sy. No. 172 & 173 Apparel Park Road Dulapally Hyderabad-500014 Andhra Pradesh India Tel.: + 91/40/2379 2190 Fax: + 91/40/2379 2223 [email protected]
N. Batta1, 2, R. K. Pigili1, L. M. K. Pallapothu1, 2, R. P. Yejella2 1 2
Bioanalytical Department, Aizant Drug Research Solutions Pvt. Ltd., Hyderabad, India University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India
Abstract
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A simple, rapid and sensitive ultra flow liquid chromatography-tandem mass spectrometry (UFLCMS/MS) assay for the determination of norgestrel in human plasma was developed using levonorgestrel D6 as an internal standard (IS). Norgestrel and IS were extracted from human plasma via liquid-liquid extraction. Chromatographic separation was achieved on a Zorbax XDB-Phenyl column under isocratic conditions. Detection was done by tandem mass spectrometry, operating in positive ion mode. The protonated precursor to product ion transitions monitored for norgestrel and IS were at m/z 313.30→245.40 and 319.00→251.30
Introduction
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Norgestrel (dl-13-beta-ethyl-17-α-ethinyl-17-βhydroxygon-4-en-3-one) is a progestin used in hormonal contraceptives. It is used for contraception, control of menstrual disorders and treatment of endometriosis. Pharmacological action includes binding of norgestrel to the progesterone and estrogen receptors. Target cells include the female reproductive tract, the mammary gland, the hypothalamus and the pituitary. Once bound to the receptor, progestin’s like norgestrel will slow the frequency of release of gonadotropin releasing hormone (GnRH) from the hypothalamus and blunt the pre-ovulatory LH (luteinizing hormone) surge. Norgestrel is a hormone that prevents pregnancy by changing the womb and cervical mucus to make it more difficult for an egg to meet sperm (fertilization) or for the fertilized egg to attach to the wall of the womb (implantation) [1–4]. Norgestrel is used for the treatment of menopausal and postmenopausal disorders it can be used alone or in combination with other hormones as an oral contraceptive [5, 6]. With the introduction
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
respectively. The method was fully validated as per current regulatory guidelines. Anticoagulant counter ion effect was also assessed with K2EDTA and K3EDTA. The method was validated with a linearity range of 304.356–50 807.337 pg/mL having run time of 2.0 min per sample. The method has shown tremendous reproducibility with intraand inter-day precision ( %CV) less than 11.0 % and intra- and inter-day accuracy less than 9.0 % of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administrating a single oral dose of 0.3 mg norgestrel tablets to healthy female volunteers and has proved to be highly reliable for the analysis of clinical samples.
of combinational drugs that too in very low doses there is a growing concern about of possible interactions from co-administered drugs and also the food-drug interactions. Many well-documented studies have been done to investigate these interactions, for example, cytochrome P450 3A4 (CYP 3A4) inhibitors, such as grape fruit juice, have been to decrease the pre-systemic elimination of norgestrel and its combinational drugs, by inhibiting metabolism. Cytochrome P450 3A4 (CYP 3A4) inducers, such as nevirapine have been reported to cause a moderate reduction in AUCinfinity, Cmax, Mean Residence Time (MRT) and t1/2 of drug in human subjects [7, 8]. To address this issue, a highly sensitive and selective bioanalytical method will be needed to accurately determine the low levels of norgestrel in human matrices. Very few analytical methods have been reported for the estimation of norgestrel by using the techniques like high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection [9, 10], gas chromatography (GC) with mass spectrometric (MS) detection [11] and radioimmunoassay (RIA) [12]. The HPLC with UV detection have the issue of sensitivities and the GC meth-
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Authors
Original Article 463
Experimental
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Materials and methods Reference standards of norgestrel (chemical purity 99.8 %, C21H28O2, MW = 312.45) and levonorgestrel D6 (chemical purity ▶ Fig. 1) were obtained from 99.6 %, C21H22O2D6, MW = 318.49) (● Clearsynth. HPLC grade acetonitrile, methanol, ammonia, methyl tert-butyl ether (tBME), water and ammonium acetate were obtained from Merck Ltd. (Mumbai, India). Blank human blood was collected with K2EDTA as well as K3EDTA anticoagulants separately from healthy volunteers. Plasma was separated by centrifugation at 4 500 rpm at 10 °C for a period of 30 min.
LC/ESI-MS/MS instrumentation and analytical conditions The liquid chromatography separation was performed using Shimadzu scientific instruments (Shimadzu Corporation: Kyoto, Japan) consisted of two LC-20AD delivery pumps, an on-line DGU-20A3 prominence solvent degasser, a SIL-HTc Shimadzu autosampler and a CBM-20A prominence column oven. Liquid chromatographic separation was achieved on Zorbax XDBPhenyl (3.5 μ, 75 × 4.6 mm) column (Agilent technologies, USA). The mobile phase used was Methanol-Acetonitrile-Ammonium Acetate (2 mM) (40:40:20, v/v/v). Flow rate was 1.00 mL/min with 70 % splitting, column and autosampler were maintained at 40 ± 5 °C and 5 ± 3 °C respectively. An injection volume of 0.015 mL was used for each analysis and runtime was 2.0 min. Samples were analyzed with API-4000 triple quadruple mass spectrometer (MDS Sciex®; Toronto, Canada) equipped with an electrospray ionization source operating in positive ion mode. Nitrogen was used as the nebulizer, auxillary, collision and curtain gases. Norgestrel and its internal standard were detected by tandem mass spectrometry using multiple reaction monitoring (MRM) of precursor-product ion transitions with 200 ms dwell time, at m/z 313.30/245.40 for norgestrel, m/z 319.00/251.30 for Levonorgestrel D6. The main working source/gas parameters of the mass spectrometer were optimized and maintained as follows: collision activated dissociation (CAD) gas, 6; curtain gas, 30; gas 1 (nebulizer gas), 50; gas 2 (heater gas), 50; turbo ion spray (IS) voltage, 5 000; source temperature, 400 °C. The com-
pound parameters like, declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were optimized and set at 62, 10, 29 and 16 V, respectively, for norgestrel and levonorgestrel D6. Data acquisition and processing were performed using Analyst version 1.4.2 software (MDS Sciex; Toronto, Canada).
Preparation of stock solution, standard and quality control samples Stock solutions of norgestrel and levonorgestrel D6 were prepared by dissolving accurately weighed standard compounds in methanol to yield a concentration of 1 mg/ml. All subsequent dilutions were made with methanol. Standard working solutions of norgestrel at concentrations of 15 217.813, 30 435.627, 126 815.112, 253 630.224, 634 075.560, 1 268 151.119, 2 032 293.460 and 2 540 366.825 pg/ml were prepared by serial dilutions. QC working solutions at concentrations of 15 314.391, 44 006.869, 144 284.817, 212 183.555, 1 060 917.774, and 2 121 835.548 pg/ml were also prepared by successively diluting the 1 mg/ml QC stock solution. The internal standard stock solution was diluted to a working concentration of 500 ng/ml. These working solutions were stored at 2–8 °C. The linearity curve was build using 8 different concentrations ranging from 304.356–50 807.337 pg/mL in human plasma.
Liquid liquid extraction (LLE) procedure Extracted sample preparation Plasma samples frozen below − 20 °C were thawed on the day of extraction at room temperature followed by vortexing to ensure homogeneity. For the determination of free norgestrel, 0.400 ml of spiked plasma was transferred to polypropylene tubes followed by 0.050 ml of IS working solution and vortex for 5 s. 0.300 mL of extraction buffer was added to all the vials and vortexed for about 60 s and then added 2.500 mL of tertiary butyl methyl ether (tBME) and vortexed for a period of 10 min by interrupting the vortexer for every 1 min. Transferred about 2.100 mL of supernatant into pre-labeled tubes and evaporated the supernatant solutions to dryness under nitrogen at 40 ± 5 °C. The dried extract was reconstituted with 150 μL of reconstitution solution and vortexed for about 1 min. Transferred appropriate volumes of the reconstituted solution into pre-labeled autosampler vials and injected 0.015 mL into LC-MS/MS through column.
Aqueous sample preparation Transferred 0.080 mL of respective spiking solutions into prelabeled tubes. 0.500 mL of 500 ng/mL ISTD WS was added to the above tubes and vortexed to mix. 0.920 mL of reconstitution solution was added and vortexed for a period of 1 min. Transferred appropriate volumes of the reconstituted solution into pre-labeled autosampler vials and injected 0.015 mL into LC-MS/ MS through column.
Method validation A full method validation was performed according to guidelines set by US FDA [17]. The validation of this procedure was performed in order to evaluate the method in terms of selectivity, sensitivity, linearity of response, accuracy, precision, recovery, matrix effect, matrix factor, ruggedness, reinjection reproducibility, effect of potential interfering drugs, day zero assessment batch, stability of analyte during both short-term sample processing and long-term storage.
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
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ods are having time consuming sample preparation and longer run times. In case of RIA it requires handling of radioactive materials, prolonged incubation and prone to cross reactivity. So here the liquid chromatography with tandem mass spectroscopy is the successor having the better sensitivity and selectivity [13, 14]. In this LC-MS/MS technique there were few methods available for norgestrel with other steroids in serum and water effluents [15, 16]. But there were no methods available for the determination of norgestrel alone in human plasma. As there are many formulations available for norgestrel alone in the market and hence we have concentrated on estimation of norgestrel in human plasma with better sensitivity and lesser runtime. This article describes a full validation of a LC-MS/MS method for quantifying norgestrel in human plasma. The sample preparation is simple, rapid procedure and no derivatization was required. This is the first validated assay to determine norgestrel alone in human plasma. Application of the method to the analysis of human plasma samples collected from healthy female volunteers, treated with norgestrel during a pharmacokinetic study is reported.
464 Original Article
Fig. 1 Chemical structures of norgestrel and levonorgestrel D6.
Norgestrel
Levonorgestrel D6
Pharmacokinetic study design The pharmacokinetic study of norgestrel was performed in healthy adult female subjects (n = 10) under both fasting and fed conditions. Blood samples were collected following oral administration of 0.3 mg tablet of norgestrel at pre-dose and 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 3.00, 3.50, 4.00, 4.50, 5.00, 6.00, 8.00, 12.00, 24.00, 36.00, 48.00, 72.00, 96.00 and 120 h post dose in K3EDTA vacutainer collection tubes (BD, Franklin, NJ, USA). The tubes were centrifuged at 4 000 rpm for 10 min and the plasma was collected. The collected plasma samples were stored below − 20 °C until analysis. Plasma samples were spiked with the IS and processed as per the extraction procedure described earlier. Along with the clinical samples, 4 sets of QC samples at low, middle and high concentration levels were assayed by distributing among the unknown samples in the analytical run. Plasma concentration-time profile of norgestrel was analyzed by non-compartmental method using WinNonlin Version 5.1 software. An incurred sample reanalysis (ISR) was performed on 30 time points each from fasting and fed healthy female volunteers. 3 points from each volunteer were taken up for ISR of which one Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
time point was Cmax, the second time point was from absorption phase above LQC and the third time point was from elimination phase above LQC. The basic objective of ISR was to confirm the initial values and to demonstrate that the assay is reproducible. The confirmation of the original results with the ISR sample is calculated as % difference. The % difference should be within 20 % for at least 67 % of the total reanalyzed incurred samples [18].
Results and Discussion
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LC-MS/MS condition optimization Optimisation was performed in both positive and negative ionization modes. But good response was achieved in positive ionization mode. Several fragment ions were observed in the product ion spectra of both norgestrel and its internal standard. Fragment ion at m/z 245.40 was chosen as product ion for norgestrel, where as fragment ion at m/z 251.30 was selected for levonorgestrel D6 as these ions presented a higher abundance, ▶ Fig. 2a, b shows the MS/MS stability and no cross-talk effect. ● spectra of norgestrel and its internal standard levonorgestrel D6.
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Fig. 2 a Product ion mass spectra [M + H] + of norgestrel. b Product ion mass spectra [M + H] + of levonorgestrel D6.
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Original Article 465
Fig. 3 Typical MRM chromatograms of norgestrel (left panel) and IS (right panel) in a human blank plasma b human plasma spiked with IS c LLOQ sample along with IS d ULOQ sample along with IS.
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
466 Original Article
Intra-day (n = 6)
Inter-day (n = 18)
of Norgestrel (pg/ml)
Mean
CV ( %)
Accuracy ( %)
Mean
CV ( %)
Accuracy ( %)
HQC(42436.711) MQC1(21218.355) MQC2(4243.671) LQC(880.137) LLOQQC(306.288)
45 528.4527 22 244.3570 4 520.6513 851.4092 299.7172
2.49 1.63 2.57 3.30 6.20
107.29 104.84 106.53 96.74 97.85
45 048.6121 21 985.8959 4 429.5492 863.2954 302.1715
2.38 1.96 2.49 3.63 10.83
106.15 103.62 104.38 98.09 98.66
Separation was attempted using various combinations of acetonitrile, methanol and buffer with varying contents of each component on different columns like C8, C18 and phenyl. Zorbax XDB-Phenyl (3.5 μ, 75 × 4.6 mm) column gave a good peak shape and response even at LLOQ level for norgestrel and IS. Mobile phase with the combination of methanol, acetronitrile and 2 mM ammonium acetate (40:40:20 V/V/V) was finalized after trying different combinations. Here methanol was included in mobile phase to increase the sensitivity and for better peak shape. The retention time of norgestrel is 1.29 min. A representative chromatogram of double blank, blank, lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ) samples ▶ Fig. 3. are shown in ●
Table 1a Precision and accuracy of norgestrel in K2EDTA.
Table 1b Precision and accuracy of norgestrel in K3EDTA. Nominal concentration
Intra-day (n = 6)
of Norgestrel (pg/ml)
Mean
CV ( %)
Accuracy ( %)
HQC(42436.711) MQC1(21218.355) MQC2(4243.671) LQC(880.137) LLOQQC(306.288)
46 546.0533 22 714.1720 4 566.6593 906.7907 335.1265
2.00 1.21 0.94 3.68 1.79
109.68 107.05 107.61 103.03 109.42
method with MRM acquisition is suitable for the selective detection of these compounds from human plasma.
Sample preparation optimization
Linearity and sensitivity
Solid Phase extraction (SPE) and Liquid-Liquid extraction (LLE) techniques are often used in the preparation of biological samples due to their ability to improve the sensitivity and robustness of the assay. LLE procedure is cost effective when compared to SPE and hence we have developed simple LLE procedure for norgestrel. Here we tried many trails with different extraction buffers and organic solvents. PH of the buffer plays a major role in the extraction procedure, so it was adjusted to 10.0 by using ammonia to get the better reproducible results. Tertiary butyl methyl ether (tBME) was chosen as organic solvent because of its better extraction efficiency.
All the 3 calibration curves analyzed during the course of validation were found to be linear for the standards concentration ranging from 304.356 to 50 807.337 pg/mL. After comparing the 2 weighting models (1/x and 1/x2), a regression equation with a weighting factor of 1/x2 of the drug to the IS concentration was found to produce the best fit for the concentration-detector response relationship for norgestrel in human plasma. The typical equation for calibration curve was y = 0.0000215x + 0.000283, r = 0.9969 in K2EDTA human plasma and y = 0.0000212x + ( − 0.000737), r = 0.9996 in K3EDTA human plasma. The sensitivity of the method evaluated by analyzing 6 replicates of LLOQ (304.356 pg/mL) samples. The % CV and % mean accuracy for norgestrel at LLOQ level was found to be 6.21 % and 97.93 % respectively.
Selection of internal standard A good internal standard must mimic the analyte during extraction process, HPLC injection and ionization variability. For LC-MS/MS analysis, use of stable isotope-labeled compounds as internal standard will have the same extraction recovery and ionization response. Hence levonorgestrel D6 has been selected as an internal standard. Extraction recovery of the internal standard was almost the same as that of norgestrel.
Method validation parameters Carryover effect The carryover effect due to the auto sampler was investigated by injecting a sequence of unextracted samples consisting of reconstitution solution, AQ ULOQ, reconstitution solution, AQ LLOQ and extracted samples containing of STD Blk, EX ULOQ, STD Blk and EX LLOQ. No significant carry over observed during this experiment.
Selectivity and chromatography The selectivity of the method was tested by analysis of blank human plasma samples from 10 different plasma lots. A representative MRM chromatogram obtained for drug free human ▶ Fig. 3a. No interfering peaks due to endogplasma is shown in ● enous species were observed at the elution time expected for norgestrel and levonorgestrel D6. This indicates that the HPLC
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
Precision and accuracy The precision ( % CV) and accuracy of the LC-MS/MS method was evaluated in K2EDTA by analyzing 6 replicates at different concentration levels corresponding to higher (HQC), middle (MQC1, MQC2), lower (LQC) and lower limit of quantification (LLOQ QC) quality control samples during the course of validation. The precision and accuracy of norgestrel in the inter-day and intra-day runs were within ± 15 % at HQC, MQC1, MQC2 and LQC concentrations and within ± 20 % at LLOQ QCs. The results are summa▶ Table 1a. rized in ● The precision and accuracy of norgestrel in K3EDTA were within ± 15 % at HQC, MQC1, MQC2 and LQC concentrations and within ± 20 % at LLOQ QCs. The results are summarized in ▶ Table 1b. ●
Recovery The % mean recoveries of norgestrel and its internal standard were determined by measuring the responses of the extracted plasma quality control samples against unextracted quality control samples at HQC, MQC1, MQC2 and LQC levels. Over all recovery and % CV at all QC levels was 78.881 and 3.64 respectively,
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Nominal concentration
Original Article 467
Matrix effect and matrix factor The matrix effect for the LC-MS/MS method was assessed in K2EDTA and K3EDTA plasma lots, by using 4 different lots of normal plasma, 1 hemolytic plasma and 1 lipidemic plasma. With each lot of plasma, samples concentration equivalent to HQC and LQC were prepared in triplicates at each level and injected. No
Table 2 Recovery of norgestrel and its internal standard. Analyte Norgestrel Levonorgestrel-D6
HQC
MQC1
81.15
76.39
MQC2
LQC
81.58
Overall mean
76.41
CV
accuracy
( %)
78.881 80.350
3.64
Table 3a Matrix effect of norgestrel in K2EDTA and K3EDTA human plasma. K2EDTA Overall mean ( %) (n = 24) CV( %) Mean accuracy ( %)
K3EDTA
HQC
LQC
HQC
LQC
44 564.9195
889.7463
45 733.0293
889.9685
3.02 101.09
1.92 107.77
2.25 101.12
1.65 105.02
Table 3b Matrix factor for analyzing norgestrel in human plasma. Matrix Lots 1 2 3 4 5 6 Mean SD CV ( %)
ISTD Normalised MF HQC
MQC1
LQC
1.00 1.00 1.01 1.02 1.00 1.01 1.008 1.0080 0.80
0.99 1.01 1.02 1.02 1.00 1.00 1.007 0.0115 1.14
0.99 1.00 1.01 0.98 0.99 0.98 0.992 0.0127 1.28
significant matrix effect was observed in all the 8 lots of human plasma in both the anticoagulants. Matrix factor was assessed by using 6 different lots (4 normal plasma, 1 hemolytic plasma and 1 lipidemic plasma) of previously screened plasma. Blank samples in duplicate for each lot in each level were processed and after extraction, evaporation the samples were spiked to achieve the concentration equivalent to HQC, MQC1 and LQC and injected. Unextracted samples concentration equivalent to HQC, MQC1 and LQC were prepared and injected. The % CV of ISTD normalized matrix factor at HQC, MQC1 and LQC samples was found to be 0.80, 1.14 and 1.28 respectively. The overall % CV of ISTD normalized matrix factor from all the 3 levels (HQC, MQC1 and LQC) was found to be 0.91 was within ≤ 15 %. The results were sum▶ Table 3a, b. marized in ●
Dilution integrity The dilution integrity of the method was evaluated by preparing the DI spiking solution (6 314 986.750 pg/mL) which is approximately equivalent to 2.5 times of the highest CC spiking solution which were spiked in the screened plasma to get a DIQC concentration (126 299.735 pg/mL). DIQC sample is further diluted by 1/5 and 1/10 times. The precision and accuracy for dilution integrity standards at 1/5 and 1/10 dilution were determined by analyzing 6 replicates samples at each dilution against calibration curve standards. The % CV for dilution integrity of 1/5 and 1/10 was found to be 1.48 and 2.17 respectively. The % mean accuracy for dilution integrity of 1/5 and 1/10 was found to be 102.43 and 98.80 respectively.
Stability studies The stabilities of norgestrel were investigated at 2 concentrations of QC samples (low and high concentrations) to cover expected conditions during analysis, storage and processing of all samples, which include the stability data from various stability exercise like in-injector, bench-top, freeze thaw, short term and long-term stability tests. Based on the stability experiments carried out during the course of validation, it was concluded that the intended analyte is stable in all the performed experiments and the stability experiments performed are within the accept▶ Table 4. ance limits. The results were summarized in ●
Table 4 Stability data of norgestrel under various conditions. Storage period and storage condition
Nominal concentration
Mean
CV ( %)
44 329.9398 878.8317 44 801.8965 858.2752 44 720.9007 844.7842 45 387.0633 853.8712 44 866.5982 852.6688 45 192.3222 846.4713 45 121.0558 861.5458
1.98 2.69 1.79 3.35 1.81 1.44 2.23 2.60 2.16 3.63 1.56 3.04 1.44 2.62
Accuracy ( %)
% Mean Stability
(pg/ml) Long term stability (100 days) Bench top stability (13 h 35 min) Auto sampler stability (73 h 33 min) Four freeze thaw cycles ( − 28 ± 5 °C) Wet Extract Stability (Room Temperature) Wet Extract Stability (2–8 °C) Dry Extract Stability ( − 28 ± 5 °C)
HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC
42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137
104.46 99.85 105.57 97.52 105.38 95.98 106.95 97.02 105.73 96.88 106.49 96.17 106.33 97.89
98.07 100.37 98.77 101.24 98.59 99.65 100.06 100.73 98.91 100.58 99.63 99.85 99.47 101.63
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which is within the acceptance limit of 20 %. The overall % mean recovery for internal standard was found to be 80.350.The ▶ Table 2. results were summarized in ●
468 Original Article
Table 5 Pharmacokinetic parameters (mean ± SD) of 0.3 mg norgestrel tablets under fasting and fed conditions. Parameters
Fasting
Fed
Tmax (h) Cmax (pg/mL) AUC0–t AUC0–∞ T1/2 (h)
1.63 ± 0.669 10 503.367 ± 3 509.2311 160 947.828 ± 72 202.9876 188 308.871 ± 80 635.1394 38.191 ± 7.2417
2.55 ± 0.832 11 102.661 ± 2 578.5036 211 820.292 ± 102 632.6718 245 154.386 ± 110 373.0512 38.219 ± 14.3452
Application to a pharmacokinetic study In order to verify the sensitivity and selectivity of this method in a real-time situation, the present method was used to test for 0.3 mg tablets of norgestrel in human plasma samples collected from healthy female volunteers (n = 10) under fasting and fed conditions. The mean plasma concentrations vs. time profiles of ▶ Fig. 4 and the mean estimated pharmanorgestrel is shown in ● cokinetic parameters derived from the plasma concentration ▶ Table 5. profile are summarized in ● Finally as per the results obtained from the ISR, it was observed that 95 % of the sample points were within ± 20 % of initial concentration value, further providing the proposed method is reproducible and suitable for pharmacokinetic evaluation of norgestrel in both fasting and fed conditions.
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
Conclusion
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A rapid, sensitive and highly selective method for the determination of norgestrel in human plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. This method can be used for analysis of large number of samples with greater precision during pharmacokinetic and bioequivalence studies. The assay method is more selective than previously described methods (HPLC, RIA) and allows for a much higher sample throughput due to the short chromatographic time (2.0 min) and simple sample preparation. As robust LC-MS/MS instrument performance was observed and hence this method is an excellent analytical option for rapid quantification of norgestrel in human plasma. The method was used successfully for the determination of plasma drug concentrations in human plasma samples under both fasting and fed conditions.
Acknowledgements
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The authors gratefully acknowledge Aizant Drug Research Solutions Pvt. Ltd., Hyderabad, India for providing necessary facilities to carry out this research work.
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Fig. 4 Mean plasma concentration-time profile of norgestrel under fasting a and fed b conditions.
Conflict of Interest
▼
We are here with declaring that, we do not have any conflicts on this research work, and we will be responsible for any conflicts raised on this manuscript.
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9 Catherine M, John K. Development of a Validated Liquid Chromatography Method for the Simultaneous Determination of Ethinyl Estradiol, Cyproterone Acetate and Norgestrel in Breast Milk Following SolidPhase Extraction. Journal of Liquid Chromatography & Related Technologies 2006; 29: 685–700 10 Gorog S, Bihari M, Csizer E et al. Estimation of impurity profiles of drugs and related materials. Part 14: the role of HPLC/diode-array UV spectroscopy in the identification of minor components (impurities, degradation products, metabolites) in various matrices. Journal of Pharmaceutical and Biomedical Analysis 1995; 14: 85–92 11 Tetsuo M, Axelson M, Sjovall J. Selective isolation procedures for GC/MS analysis of ethynyl steroids in biological material. Journal of Steroid Biochemistry 1980; 13: 847–860 12 Warren RJ, Fotherby K. Radioimmunoassay of synthetic Progestogens, Norethisterone and Norgestrel. Journal of Endocrinology 1974; 62: 605–618 13 Brunis AP. Atmospheric-pressure-ionization mass spectrometry: I. Instrumentation and ionization techniques. Trends in Analytical Chemistry 1994; 13: 81–90 14 Muck W. Quantitative analysis of pharmacokinetic study samples by liquid chromatography coupled to tandem mass spectrometry (LCMS/MS). Pharmazie 1999; 54: 639–644 15 Wu Z, Zhang C, Yang C et al. Simultaneous quantitative determination of norgestrel and progesterone in human serum by high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization. Analyst 2000; 125: 2201–2205 16 Sapozhnikova Y, Hedgespeth M, Wirth E et al. Analysis of selected natural and synthetic hormones by LC-MS-MS using the US EPA method 1694. Analytical Methods 2011; 3: 1079–1086 17 Guidance for Industry. Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research. Rockville, MD: 2001 Available from: http://www.fda.gov/CVM 18 Viswanathan CT, Bansal S, Booth B et al. Workshop/conference report – quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. AAPS Journal 2007; 9: E30–E42
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Original Article 469
A Simple, Rapid and Sensitive UFLC-MS/MS Method for the Quantification of Oral Contraceptive Norgestrel in Human Plasma and its Pharmacokinetic Applications
Affiliations
Key words ▶ norgestrel ● ▶ oral contraceptive ● ▶ UFLC-MS/MS ● ▶ method validation ● ▶ pharmacokinetics ●
received 29.10.2013 accepted 15.11.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1361164 Published online: December 5, 2013 Drug Res 2014; 64: 462–469 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence L. M. K. Pallapothu Deputy Director, Bioanalytical Aizant Drug Research Solutions (P) Ltd. Sy. No. 172 & 173 Apparel Park Road Dulapally Hyderabad-500014 Andhra Pradesh India Tel.: + 91/40/2379 2190 Fax: + 91/40/2379 2223 [email protected]
N. Batta1, 2, R. K. Pigili1, L. M. K. Pallapothu1, 2, R. P. Yejella2 1 2
Bioanalytical Department, Aizant Drug Research Solutions Pvt. Ltd., Hyderabad, India University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India
Abstract
▼
A simple, rapid and sensitive ultra flow liquid chromatography-tandem mass spectrometry (UFLCMS/MS) assay for the determination of norgestrel in human plasma was developed using levonorgestrel D6 as an internal standard (IS). Norgestrel and IS were extracted from human plasma via liquid-liquid extraction. Chromatographic separation was achieved on a Zorbax XDB-Phenyl column under isocratic conditions. Detection was done by tandem mass spectrometry, operating in positive ion mode. The protonated precursor to product ion transitions monitored for norgestrel and IS were at m/z 313.30→245.40 and 319.00→251.30
Introduction
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Norgestrel (dl-13-beta-ethyl-17-α-ethinyl-17-βhydroxygon-4-en-3-one) is a progestin used in hormonal contraceptives. It is used for contraception, control of menstrual disorders and treatment of endometriosis. Pharmacological action includes binding of norgestrel to the progesterone and estrogen receptors. Target cells include the female reproductive tract, the mammary gland, the hypothalamus and the pituitary. Once bound to the receptor, progestin’s like norgestrel will slow the frequency of release of gonadotropin releasing hormone (GnRH) from the hypothalamus and blunt the pre-ovulatory LH (luteinizing hormone) surge. Norgestrel is a hormone that prevents pregnancy by changing the womb and cervical mucus to make it more difficult for an egg to meet sperm (fertilization) or for the fertilized egg to attach to the wall of the womb (implantation) [1–4]. Norgestrel is used for the treatment of menopausal and postmenopausal disorders it can be used alone or in combination with other hormones as an oral contraceptive [5, 6]. With the introduction
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
respectively. The method was fully validated as per current regulatory guidelines. Anticoagulant counter ion effect was also assessed with K2EDTA and K3EDTA. The method was validated with a linearity range of 304.356–50 807.337 pg/mL having run time of 2.0 min per sample. The method has shown tremendous reproducibility with intraand inter-day precision ( %CV) less than 11.0 % and intra- and inter-day accuracy less than 9.0 % of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administrating a single oral dose of 0.3 mg norgestrel tablets to healthy female volunteers and has proved to be highly reliable for the analysis of clinical samples.
of combinational drugs that too in very low doses there is a growing concern about of possible interactions from co-administered drugs and also the food-drug interactions. Many well-documented studies have been done to investigate these interactions, for example, cytochrome P450 3A4 (CYP 3A4) inhibitors, such as grape fruit juice, have been to decrease the pre-systemic elimination of norgestrel and its combinational drugs, by inhibiting metabolism. Cytochrome P450 3A4 (CYP 3A4) inducers, such as nevirapine have been reported to cause a moderate reduction in AUCinfinity, Cmax, Mean Residence Time (MRT) and t1/2 of drug in human subjects [7, 8]. To address this issue, a highly sensitive and selective bioanalytical method will be needed to accurately determine the low levels of norgestrel in human matrices. Very few analytical methods have been reported for the estimation of norgestrel by using the techniques like high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection [9, 10], gas chromatography (GC) with mass spectrometric (MS) detection [11] and radioimmunoassay (RIA) [12]. The HPLC with UV detection have the issue of sensitivities and the GC meth-
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Authors
Original Article 463
Experimental
▼
Materials and methods Reference standards of norgestrel (chemical purity 99.8 %, C21H28O2, MW = 312.45) and levonorgestrel D6 (chemical purity ▶ Fig. 1) were obtained from 99.6 %, C21H22O2D6, MW = 318.49) (● Clearsynth. HPLC grade acetonitrile, methanol, ammonia, methyl tert-butyl ether (tBME), water and ammonium acetate were obtained from Merck Ltd. (Mumbai, India). Blank human blood was collected with K2EDTA as well as K3EDTA anticoagulants separately from healthy volunteers. Plasma was separated by centrifugation at 4 500 rpm at 10 °C for a period of 30 min.
LC/ESI-MS/MS instrumentation and analytical conditions The liquid chromatography separation was performed using Shimadzu scientific instruments (Shimadzu Corporation: Kyoto, Japan) consisted of two LC-20AD delivery pumps, an on-line DGU-20A3 prominence solvent degasser, a SIL-HTc Shimadzu autosampler and a CBM-20A prominence column oven. Liquid chromatographic separation was achieved on Zorbax XDBPhenyl (3.5 μ, 75 × 4.6 mm) column (Agilent technologies, USA). The mobile phase used was Methanol-Acetonitrile-Ammonium Acetate (2 mM) (40:40:20, v/v/v). Flow rate was 1.00 mL/min with 70 % splitting, column and autosampler were maintained at 40 ± 5 °C and 5 ± 3 °C respectively. An injection volume of 0.015 mL was used for each analysis and runtime was 2.0 min. Samples were analyzed with API-4000 triple quadruple mass spectrometer (MDS Sciex®; Toronto, Canada) equipped with an electrospray ionization source operating in positive ion mode. Nitrogen was used as the nebulizer, auxillary, collision and curtain gases. Norgestrel and its internal standard were detected by tandem mass spectrometry using multiple reaction monitoring (MRM) of precursor-product ion transitions with 200 ms dwell time, at m/z 313.30/245.40 for norgestrel, m/z 319.00/251.30 for Levonorgestrel D6. The main working source/gas parameters of the mass spectrometer were optimized and maintained as follows: collision activated dissociation (CAD) gas, 6; curtain gas, 30; gas 1 (nebulizer gas), 50; gas 2 (heater gas), 50; turbo ion spray (IS) voltage, 5 000; source temperature, 400 °C. The com-
pound parameters like, declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were optimized and set at 62, 10, 29 and 16 V, respectively, for norgestrel and levonorgestrel D6. Data acquisition and processing were performed using Analyst version 1.4.2 software (MDS Sciex; Toronto, Canada).
Preparation of stock solution, standard and quality control samples Stock solutions of norgestrel and levonorgestrel D6 were prepared by dissolving accurately weighed standard compounds in methanol to yield a concentration of 1 mg/ml. All subsequent dilutions were made with methanol. Standard working solutions of norgestrel at concentrations of 15 217.813, 30 435.627, 126 815.112, 253 630.224, 634 075.560, 1 268 151.119, 2 032 293.460 and 2 540 366.825 pg/ml were prepared by serial dilutions. QC working solutions at concentrations of 15 314.391, 44 006.869, 144 284.817, 212 183.555, 1 060 917.774, and 2 121 835.548 pg/ml were also prepared by successively diluting the 1 mg/ml QC stock solution. The internal standard stock solution was diluted to a working concentration of 500 ng/ml. These working solutions were stored at 2–8 °C. The linearity curve was build using 8 different concentrations ranging from 304.356–50 807.337 pg/mL in human plasma.
Liquid liquid extraction (LLE) procedure Extracted sample preparation Plasma samples frozen below − 20 °C were thawed on the day of extraction at room temperature followed by vortexing to ensure homogeneity. For the determination of free norgestrel, 0.400 ml of spiked plasma was transferred to polypropylene tubes followed by 0.050 ml of IS working solution and vortex for 5 s. 0.300 mL of extraction buffer was added to all the vials and vortexed for about 60 s and then added 2.500 mL of tertiary butyl methyl ether (tBME) and vortexed for a period of 10 min by interrupting the vortexer for every 1 min. Transferred about 2.100 mL of supernatant into pre-labeled tubes and evaporated the supernatant solutions to dryness under nitrogen at 40 ± 5 °C. The dried extract was reconstituted with 150 μL of reconstitution solution and vortexed for about 1 min. Transferred appropriate volumes of the reconstituted solution into pre-labeled autosampler vials and injected 0.015 mL into LC-MS/MS through column.
Aqueous sample preparation Transferred 0.080 mL of respective spiking solutions into prelabeled tubes. 0.500 mL of 500 ng/mL ISTD WS was added to the above tubes and vortexed to mix. 0.920 mL of reconstitution solution was added and vortexed for a period of 1 min. Transferred appropriate volumes of the reconstituted solution into pre-labeled autosampler vials and injected 0.015 mL into LC-MS/ MS through column.
Method validation A full method validation was performed according to guidelines set by US FDA [17]. The validation of this procedure was performed in order to evaluate the method in terms of selectivity, sensitivity, linearity of response, accuracy, precision, recovery, matrix effect, matrix factor, ruggedness, reinjection reproducibility, effect of potential interfering drugs, day zero assessment batch, stability of analyte during both short-term sample processing and long-term storage.
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ods are having time consuming sample preparation and longer run times. In case of RIA it requires handling of radioactive materials, prolonged incubation and prone to cross reactivity. So here the liquid chromatography with tandem mass spectroscopy is the successor having the better sensitivity and selectivity [13, 14]. In this LC-MS/MS technique there were few methods available for norgestrel with other steroids in serum and water effluents [15, 16]. But there were no methods available for the determination of norgestrel alone in human plasma. As there are many formulations available for norgestrel alone in the market and hence we have concentrated on estimation of norgestrel in human plasma with better sensitivity and lesser runtime. This article describes a full validation of a LC-MS/MS method for quantifying norgestrel in human plasma. The sample preparation is simple, rapid procedure and no derivatization was required. This is the first validated assay to determine norgestrel alone in human plasma. Application of the method to the analysis of human plasma samples collected from healthy female volunteers, treated with norgestrel during a pharmacokinetic study is reported.
464 Original Article
Fig. 1 Chemical structures of norgestrel and levonorgestrel D6.
Norgestrel
Levonorgestrel D6
Pharmacokinetic study design The pharmacokinetic study of norgestrel was performed in healthy adult female subjects (n = 10) under both fasting and fed conditions. Blood samples were collected following oral administration of 0.3 mg tablet of norgestrel at pre-dose and 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 3.00, 3.50, 4.00, 4.50, 5.00, 6.00, 8.00, 12.00, 24.00, 36.00, 48.00, 72.00, 96.00 and 120 h post dose in K3EDTA vacutainer collection tubes (BD, Franklin, NJ, USA). The tubes were centrifuged at 4 000 rpm for 10 min and the plasma was collected. The collected plasma samples were stored below − 20 °C until analysis. Plasma samples were spiked with the IS and processed as per the extraction procedure described earlier. Along with the clinical samples, 4 sets of QC samples at low, middle and high concentration levels were assayed by distributing among the unknown samples in the analytical run. Plasma concentration-time profile of norgestrel was analyzed by non-compartmental method using WinNonlin Version 5.1 software. An incurred sample reanalysis (ISR) was performed on 30 time points each from fasting and fed healthy female volunteers. 3 points from each volunteer were taken up for ISR of which one Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
time point was Cmax, the second time point was from absorption phase above LQC and the third time point was from elimination phase above LQC. The basic objective of ISR was to confirm the initial values and to demonstrate that the assay is reproducible. The confirmation of the original results with the ISR sample is calculated as % difference. The % difference should be within 20 % for at least 67 % of the total reanalyzed incurred samples [18].
Results and Discussion
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LC-MS/MS condition optimization Optimisation was performed in both positive and negative ionization modes. But good response was achieved in positive ionization mode. Several fragment ions were observed in the product ion spectra of both norgestrel and its internal standard. Fragment ion at m/z 245.40 was chosen as product ion for norgestrel, where as fragment ion at m/z 251.30 was selected for levonorgestrel D6 as these ions presented a higher abundance, ▶ Fig. 2a, b shows the MS/MS stability and no cross-talk effect. ● spectra of norgestrel and its internal standard levonorgestrel D6.
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Fig. 2 a Product ion mass spectra [M + H] + of norgestrel. b Product ion mass spectra [M + H] + of levonorgestrel D6.
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Original Article 465
Fig. 3 Typical MRM chromatograms of norgestrel (left panel) and IS (right panel) in a human blank plasma b human plasma spiked with IS c LLOQ sample along with IS d ULOQ sample along with IS.
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
466 Original Article
Intra-day (n = 6)
Inter-day (n = 18)
of Norgestrel (pg/ml)
Mean
CV ( %)
Accuracy ( %)
Mean
CV ( %)
Accuracy ( %)
HQC(42436.711) MQC1(21218.355) MQC2(4243.671) LQC(880.137) LLOQQC(306.288)
45 528.4527 22 244.3570 4 520.6513 851.4092 299.7172
2.49 1.63 2.57 3.30 6.20
107.29 104.84 106.53 96.74 97.85
45 048.6121 21 985.8959 4 429.5492 863.2954 302.1715
2.38 1.96 2.49 3.63 10.83
106.15 103.62 104.38 98.09 98.66
Separation was attempted using various combinations of acetonitrile, methanol and buffer with varying contents of each component on different columns like C8, C18 and phenyl. Zorbax XDB-Phenyl (3.5 μ, 75 × 4.6 mm) column gave a good peak shape and response even at LLOQ level for norgestrel and IS. Mobile phase with the combination of methanol, acetronitrile and 2 mM ammonium acetate (40:40:20 V/V/V) was finalized after trying different combinations. Here methanol was included in mobile phase to increase the sensitivity and for better peak shape. The retention time of norgestrel is 1.29 min. A representative chromatogram of double blank, blank, lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ) samples ▶ Fig. 3. are shown in ●
Table 1a Precision and accuracy of norgestrel in K2EDTA.
Table 1b Precision and accuracy of norgestrel in K3EDTA. Nominal concentration
Intra-day (n = 6)
of Norgestrel (pg/ml)
Mean
CV ( %)
Accuracy ( %)
HQC(42436.711) MQC1(21218.355) MQC2(4243.671) LQC(880.137) LLOQQC(306.288)
46 546.0533 22 714.1720 4 566.6593 906.7907 335.1265
2.00 1.21 0.94 3.68 1.79
109.68 107.05 107.61 103.03 109.42
method with MRM acquisition is suitable for the selective detection of these compounds from human plasma.
Sample preparation optimization
Linearity and sensitivity
Solid Phase extraction (SPE) and Liquid-Liquid extraction (LLE) techniques are often used in the preparation of biological samples due to their ability to improve the sensitivity and robustness of the assay. LLE procedure is cost effective when compared to SPE and hence we have developed simple LLE procedure for norgestrel. Here we tried many trails with different extraction buffers and organic solvents. PH of the buffer plays a major role in the extraction procedure, so it was adjusted to 10.0 by using ammonia to get the better reproducible results. Tertiary butyl methyl ether (tBME) was chosen as organic solvent because of its better extraction efficiency.
All the 3 calibration curves analyzed during the course of validation were found to be linear for the standards concentration ranging from 304.356 to 50 807.337 pg/mL. After comparing the 2 weighting models (1/x and 1/x2), a regression equation with a weighting factor of 1/x2 of the drug to the IS concentration was found to produce the best fit for the concentration-detector response relationship for norgestrel in human plasma. The typical equation for calibration curve was y = 0.0000215x + 0.000283, r = 0.9969 in K2EDTA human plasma and y = 0.0000212x + ( − 0.000737), r = 0.9996 in K3EDTA human plasma. The sensitivity of the method evaluated by analyzing 6 replicates of LLOQ (304.356 pg/mL) samples. The % CV and % mean accuracy for norgestrel at LLOQ level was found to be 6.21 % and 97.93 % respectively.
Selection of internal standard A good internal standard must mimic the analyte during extraction process, HPLC injection and ionization variability. For LC-MS/MS analysis, use of stable isotope-labeled compounds as internal standard will have the same extraction recovery and ionization response. Hence levonorgestrel D6 has been selected as an internal standard. Extraction recovery of the internal standard was almost the same as that of norgestrel.
Method validation parameters Carryover effect The carryover effect due to the auto sampler was investigated by injecting a sequence of unextracted samples consisting of reconstitution solution, AQ ULOQ, reconstitution solution, AQ LLOQ and extracted samples containing of STD Blk, EX ULOQ, STD Blk and EX LLOQ. No significant carry over observed during this experiment.
Selectivity and chromatography The selectivity of the method was tested by analysis of blank human plasma samples from 10 different plasma lots. A representative MRM chromatogram obtained for drug free human ▶ Fig. 3a. No interfering peaks due to endogplasma is shown in ● enous species were observed at the elution time expected for norgestrel and levonorgestrel D6. This indicates that the HPLC
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
Precision and accuracy The precision ( % CV) and accuracy of the LC-MS/MS method was evaluated in K2EDTA by analyzing 6 replicates at different concentration levels corresponding to higher (HQC), middle (MQC1, MQC2), lower (LQC) and lower limit of quantification (LLOQ QC) quality control samples during the course of validation. The precision and accuracy of norgestrel in the inter-day and intra-day runs were within ± 15 % at HQC, MQC1, MQC2 and LQC concentrations and within ± 20 % at LLOQ QCs. The results are summa▶ Table 1a. rized in ● The precision and accuracy of norgestrel in K3EDTA were within ± 15 % at HQC, MQC1, MQC2 and LQC concentrations and within ± 20 % at LLOQ QCs. The results are summarized in ▶ Table 1b. ●
Recovery The % mean recoveries of norgestrel and its internal standard were determined by measuring the responses of the extracted plasma quality control samples against unextracted quality control samples at HQC, MQC1, MQC2 and LQC levels. Over all recovery and % CV at all QC levels was 78.881 and 3.64 respectively,
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Nominal concentration
Original Article 467
Matrix effect and matrix factor The matrix effect for the LC-MS/MS method was assessed in K2EDTA and K3EDTA plasma lots, by using 4 different lots of normal plasma, 1 hemolytic plasma and 1 lipidemic plasma. With each lot of plasma, samples concentration equivalent to HQC and LQC were prepared in triplicates at each level and injected. No
Table 2 Recovery of norgestrel and its internal standard. Analyte Norgestrel Levonorgestrel-D6
HQC
MQC1
81.15
76.39
MQC2
LQC
81.58
Overall mean
76.41
CV
accuracy
( %)
78.881 80.350
3.64
Table 3a Matrix effect of norgestrel in K2EDTA and K3EDTA human plasma. K2EDTA Overall mean ( %) (n = 24) CV( %) Mean accuracy ( %)
K3EDTA
HQC
LQC
HQC
LQC
44 564.9195
889.7463
45 733.0293
889.9685
3.02 101.09
1.92 107.77
2.25 101.12
1.65 105.02
Table 3b Matrix factor for analyzing norgestrel in human plasma. Matrix Lots 1 2 3 4 5 6 Mean SD CV ( %)
ISTD Normalised MF HQC
MQC1
LQC
1.00 1.00 1.01 1.02 1.00 1.01 1.008 1.0080 0.80
0.99 1.01 1.02 1.02 1.00 1.00 1.007 0.0115 1.14
0.99 1.00 1.01 0.98 0.99 0.98 0.992 0.0127 1.28
significant matrix effect was observed in all the 8 lots of human plasma in both the anticoagulants. Matrix factor was assessed by using 6 different lots (4 normal plasma, 1 hemolytic plasma and 1 lipidemic plasma) of previously screened plasma. Blank samples in duplicate for each lot in each level were processed and after extraction, evaporation the samples were spiked to achieve the concentration equivalent to HQC, MQC1 and LQC and injected. Unextracted samples concentration equivalent to HQC, MQC1 and LQC were prepared and injected. The % CV of ISTD normalized matrix factor at HQC, MQC1 and LQC samples was found to be 0.80, 1.14 and 1.28 respectively. The overall % CV of ISTD normalized matrix factor from all the 3 levels (HQC, MQC1 and LQC) was found to be 0.91 was within ≤ 15 %. The results were sum▶ Table 3a, b. marized in ●
Dilution integrity The dilution integrity of the method was evaluated by preparing the DI spiking solution (6 314 986.750 pg/mL) which is approximately equivalent to 2.5 times of the highest CC spiking solution which were spiked in the screened plasma to get a DIQC concentration (126 299.735 pg/mL). DIQC sample is further diluted by 1/5 and 1/10 times. The precision and accuracy for dilution integrity standards at 1/5 and 1/10 dilution were determined by analyzing 6 replicates samples at each dilution against calibration curve standards. The % CV for dilution integrity of 1/5 and 1/10 was found to be 1.48 and 2.17 respectively. The % mean accuracy for dilution integrity of 1/5 and 1/10 was found to be 102.43 and 98.80 respectively.
Stability studies The stabilities of norgestrel were investigated at 2 concentrations of QC samples (low and high concentrations) to cover expected conditions during analysis, storage and processing of all samples, which include the stability data from various stability exercise like in-injector, bench-top, freeze thaw, short term and long-term stability tests. Based on the stability experiments carried out during the course of validation, it was concluded that the intended analyte is stable in all the performed experiments and the stability experiments performed are within the accept▶ Table 4. ance limits. The results were summarized in ●
Table 4 Stability data of norgestrel under various conditions. Storage period and storage condition
Nominal concentration
Mean
CV ( %)
44 329.9398 878.8317 44 801.8965 858.2752 44 720.9007 844.7842 45 387.0633 853.8712 44 866.5982 852.6688 45 192.3222 846.4713 45 121.0558 861.5458
1.98 2.69 1.79 3.35 1.81 1.44 2.23 2.60 2.16 3.63 1.56 3.04 1.44 2.62
Accuracy ( %)
% Mean Stability
(pg/ml) Long term stability (100 days) Bench top stability (13 h 35 min) Auto sampler stability (73 h 33 min) Four freeze thaw cycles ( − 28 ± 5 °C) Wet Extract Stability (Room Temperature) Wet Extract Stability (2–8 °C) Dry Extract Stability ( − 28 ± 5 °C)
HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC
42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137 42 436.711 880.137
104.46 99.85 105.57 97.52 105.38 95.98 106.95 97.02 105.73 96.88 106.49 96.17 106.33 97.89
98.07 100.37 98.77 101.24 98.59 99.65 100.06 100.73 98.91 100.58 99.63 99.85 99.47 101.63
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which is within the acceptance limit of 20 %. The overall % mean recovery for internal standard was found to be 80.350.The ▶ Table 2. results were summarized in ●
468 Original Article
Table 5 Pharmacokinetic parameters (mean ± SD) of 0.3 mg norgestrel tablets under fasting and fed conditions. Parameters
Fasting
Fed
Tmax (h) Cmax (pg/mL) AUC0–t AUC0–∞ T1/2 (h)
1.63 ± 0.669 10 503.367 ± 3 509.2311 160 947.828 ± 72 202.9876 188 308.871 ± 80 635.1394 38.191 ± 7.2417
2.55 ± 0.832 11 102.661 ± 2 578.5036 211 820.292 ± 102 632.6718 245 154.386 ± 110 373.0512 38.219 ± 14.3452
Application to a pharmacokinetic study In order to verify the sensitivity and selectivity of this method in a real-time situation, the present method was used to test for 0.3 mg tablets of norgestrel in human plasma samples collected from healthy female volunteers (n = 10) under fasting and fed conditions. The mean plasma concentrations vs. time profiles of ▶ Fig. 4 and the mean estimated pharmanorgestrel is shown in ● cokinetic parameters derived from the plasma concentration ▶ Table 5. profile are summarized in ● Finally as per the results obtained from the ISR, it was observed that 95 % of the sample points were within ± 20 % of initial concentration value, further providing the proposed method is reproducible and suitable for pharmacokinetic evaluation of norgestrel in both fasting and fed conditions.
Batta N et al. Quantification of Norgestrel … Drug Res 2014; 64: 462–469
Conclusion
▼
A rapid, sensitive and highly selective method for the determination of norgestrel in human plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. This method can be used for analysis of large number of samples with greater precision during pharmacokinetic and bioequivalence studies. The assay method is more selective than previously described methods (HPLC, RIA) and allows for a much higher sample throughput due to the short chromatographic time (2.0 min) and simple sample preparation. As robust LC-MS/MS instrument performance was observed and hence this method is an excellent analytical option for rapid quantification of norgestrel in human plasma. The method was used successfully for the determination of plasma drug concentrations in human plasma samples under both fasting and fed conditions.
Acknowledgements
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The authors gratefully acknowledge Aizant Drug Research Solutions Pvt. Ltd., Hyderabad, India for providing necessary facilities to carry out this research work.
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Fig. 4 Mean plasma concentration-time profile of norgestrel under fasting a and fed b conditions.
Conflict of Interest
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We are here with declaring that, we do not have any conflicts on this research work, and we will be responsible for any conflicts raised on this manuscript.
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