Determination of Norgestimate and Ethinyl Estradiol in Tablets by High-Performance Liquid Chromatography

Alton K.B., Hetyei N.S., Shaw C., Patrick J.E.

The metabolism of norgestimate (ORF-10131; 14C-d-13-ethyl-17-acetoxy-18,19-dinor-17 alpha-pregn-4-en-20- yn -3-oxime) was studied in humans. Compound labeled with carbon-14 in the 17 alpha-ethynyl group was administered to four female subjects. An average of 46.8 percent of the administered radioactivity was excreted in the urine and 36.8 percent in the feces over a two-week collection period. About 12 percent of the urinary radioactivity represented freely extractable metabolites and another 57 percent consisted of extractable material released by enzyme hydrolysis. The ethynylated metabolites of norgestimate were separated from endogenous compounds and non- ethynylated metabolites by silver- sulfoethyl cellulose column chromatography. Metabolites were subsequently isolated by high performance liquid chromatography and thin layer chromatography. The identification of five urinary metabolites was accomplished by combined gas-liquid chromatography/mass spectrometry. These metabolites include norgestrel, 16 beta- hydroxynorgestrel , 2 alpha- hydroxynorgestrel , 3 alpha, 5 beta- tetrahydronorgestrel , and a fifth trihydroxylated metabolite of undetermined stereochemical configuration; 3,16-dihydroxy-5- tetrahydronorgestrel .

Strusiak S.H., Hoogerheide J.G., Gardner M.S.

A rapid, reproducible high-performance liquid chromatographic system for the determination of ethinyl estradiol in solid dosage forms consisting of a reversed-phase column with a mobile phase of 0.05 M aqueous KH2PO4-methyl alcohol (2:3) and fluorescence detection has been developed. This stability-indicating method is applicable to tablets containing ethinyl estradiol alone or in combination with methyltestosterone and progesterones. The procedure has been used for the determination of ethinyl estradiol in single tablets, stability samples, and dissolution medium. Recovery of drug substance added to placebo was from 97.3 to 101.5% in stability and single-tablet assays, and 95.4 to 102.2% in dissolution assays. Reproducibility studies gave relative standard deviations of 0.4-2.2%.

Carignan G., Lodge B.A., Skakum W.

A procedure is described for the assay of ethynodiol diacetate and ethinyl estradiol/mestranol by HPLC using two UV detectors at 210 and 280 nm. The system was acetonitrile 38% (v/v) in water as mobile phase on a 250 x 3.2-mm i.d. RP-2 column, with butylated hydroxytoluene as the internal standard. There was greater than 99% recovery from synthetic preparations and the coefficient of variation was greater than 2.0% for formulations.

Gluck J.A., Shek E.

A specific and sensitive analytical method is described for the simultaneous determination of ethinylestradiol and norethisterone in a capsule formulation. These steroids, commonly used in oral contraceptives, were extracted from the capsules with acetonitrile and tetrahydrofuran. The steroids were then quantitated with a high performance liquid chromatograph using a ODS reversed-phase column and a ternary solvent system of water, acetonitrile, and tetrahydrofuran as the mobile phase. Several solvent systems for the mobile phase were examined using various C18 columns. The k' values for several steroids are reported, together with column performance parameters. It was found that columns from different manufacturers had significantly different behaviors with respect to separation parameters for these steroids.This is a description of the use of a reversed-phase HPLC (high performance liquid chromatography) system for the simultaneous analysis of both steroids, NET (norethisterone) and EE (ethinylestradiol), in a capsule form of combined OCs (oral contraceptives). The method is seen to provide a specific, quick, and accurate assay of the 2 most commonly used steroids. These 2 steriods were extracted from the capsules and quantitated with a HPLC using a bonded octadecylsilane reversed-phase column and a ternary solvent system of water, acetonitrile, and tetrahydrofuran as the mobile phase. Columns from different manufacturers behaved differently with respect to separation parameters for the 2 steroids. 2 problems inherent in the analytic method are: 1) the low ultraviolet molar absorptivity of EE; and 2) the low proportion of EE compared to NET. The described procedure can also be used for analysis of other forms of the combined steroids, e.g., tablets.

Patthy M., Tomori É.

High-performance liquid chromatographic (HPLC) and gas-liquid chromatographic (GLC) separations of some steroidal α,β-unsaturated ketoximes, intermediates in the synthesis of the oral contraceptive norgestrel, were investigated. When GLC (trimethylsilyl derivatives) or reversed-phase HPLC was used, the syn- and anti-isomers were not resolved. However, with the syn/anti ratio changing markedly in the samples, these methods (especially reversed-phase HPLC) proved to be less accurate because the physical properties relevant to detection were different for the isomers examined. Improved accuracy can be achieved by using normal-phase HPLC and taking the corresponding isolated syn- and anti-isomers as reference standards.

Cotter M.L., Levine S.D., Mallory R., Shaw C.

Bagon K.R., Hammond E.W.

A reversed-phase chromatographic system is used to separate 17α-ethinyloestradiol-1,3,5(10)-triene-3,17β-diol (ethinyloestradiol) from structurally related steroidal compounds. A direct and highly sensitive method of assay is given for uncoated tablets containing ethinyloestradiol, as used for contraception. The method is modified for sugar-coated tablets used for relief of menopause symptoms and is sufficiently sensitive to detect ethinyloestradiol if it is present with other steroids as a contaminant.

Huettemann R.E., Shroff A.P.

A high pressure liquid chromatographic determination of a steroidal oxime in the pharmaceutical dosage form is reported. The reversed phase liquid-liquid partition chromatography employing ODS-Permaphase as support allows thirty to forty individual tablets to be analyzed per hour at a dose level of 50 micrograms to 1 milligram per tablet. The method is simple, rapid, accurate and sensitive.

Huettemann R.E., Shroff A.P.

The conversion of 17α-acetoxy-6αmethyl-4-pregnen-3,20-dione 3-oxime to the corresponding diketone in acidic media was found to be a first-order reaction at 37°. The effects of incorporating ester groupings at the oxime function or at the C-17 position and modifications in ring B were also investigated. Only the length of the ester chain at the oxime function had a profound effect on the rate constant. From these observations, it is proposed that two competing mechanisms of hydrolysis are involved for the oxime esters.

Adhikari R.P., Mohamed Sheik Tharik A., Meyyanathan S.N.

Abstract In this review on the forced degradation studies on anti-epileptic drugs and the development of validated stability-indicating assay methods for drug substances and products at a condition more severe than accelerated condition (i.e. 40 ± 2°C, 75 ± 5% relative humidity), the drug substance and drug product undergo degradation is known as forced or stress degradation. To know about the impurities developed during the storage of drug products in various environmental conditions. The limit of degradation allowable is 5–20%. More than 20% of degradation is abnormal and must be investigated. Any regulatory guidelines do not mention the pH conditions for acid or base hydrolysis, the temperature for thermal degradation or the concentration of the oxidation agent. Only International Conference on Harmonization (ICH) guidelines Q1B photostability stability and states that light sources must be a combination of UV and visible light. The shortcomings of mentioned techniques with appreciation to regulatory necessities are highlighted. A systematic method for the forced degradation studies on anti-epileptic drugs such as “Topiramate, Vigabatrin, Lacosamide, Tiagabine, Levetiracetam and Zonisamide” is discussed. This review helps researchers to get an idea about stability-indicating methods of development and validation for newer antiepileptic drugs and the characteristics of drug products that degrade under specific degradation conditions.

Habib I.H., Rizk M.S., Sultan M., Mohamed D., Tony R.M.

Cathodic voltammetric behaviors of drospirenone and ethinylestradiol were used for the simultaneous determination of both drugs in bulk and in pharmaceutical formulation (Yasmin® tablets) without the interference of excipients. The determinations were made on hanging mercury dropping electrode using square-wave technique in a voltammetric cell containing 10 mL of 0.04 mole/L Britton-Robinson. After every aliquot addition, the solution was stirred for 10 s at 1000 rpm, rested for 10 s then square wave voltammetry mode was ramped from +100 to -1700 mV with scan rate of 100 mV/s, pulse amplitude of 50 mV and measurement time of 5 ms. Several factors such as pH, type of supporting electrolyte, pulse amplitude and scan rate were studied to optimize the condition for voltammetric determination of these drugs. With optimized experimental parameters, a good linearity was obtained for both drugs over a range of 1.36×10-6 to 1.91×10-7 mole/L and 6.75×10-8 to 6.07×10-7 mol/L of drospirenone and ethinylestradiol, respectively. Characterization of the proposed method was done according to International Conference on Harmonization, Q2B: Validation of Analytical procedures. The proposed method was statistically compared with the reference method and the results revealed no significant difference regarding accuracy and precision.

Mantena B.P., Rao S.V., Rao K.M., Ramakrishna K., Vittal S.P.

This paper describes a strategic development and validation of stability-indicating high-performance liquid chromatography (HPLC) method for the determination of potential impurities present in highly potent and low dose combination drug product of Norgestimate and Ethinyl estradiol tablets. Effective and faster separation of impurities from the drug products was achieved on a sub-2 µm fused core particle C8 column (150 mm × 4.6 mm i.d.) using water and acetonitrile as mobile phase in gradient pump mode. Flow rate was selected 1.0 mL/min with a detection quantification wavelength of 230 nm. The calibration curve obtained from linearity data (r > 0.995) in the relevant ranges (up to 150% of the expected concentration of the analytes in the test concentration), accuracy (>95% recovery for the impurities of respective analytes), method precision and intermediate precision (RSD 

Arsova-Sarafinovska Z., Ugrinova L., Starkoska K., Djordjev D., Dimitrovska A.

Oral contraceptives are pharmaceutical formulations containing an estrogen in a small amount and a synthetic progestin in 5-30 times bigger amount. A sensitive, accurate and rapid method for determination of active compounds is required. We have developed HPLC methods for determination of ethinylestradiol (EED) and levonorgestrel (LNG) in commercially available tablets. Chromatographic separation was performed on a Purospher® STAR RP-18e reversed-phase column (150 X 4.0 mm I.D.; particle size 5 µm) in an isocratic mode with a mobile phase constituted of 47% acetonitrile: 53% water (V/V) for both methods. The elution was carried out at a flow rate of 1.50 ml /min. All analyses were performed at room temperature (24 +/- 2°C). In the HPLC method with UV detection (internal standard method) both compounds were detected at 215 nm. Drospirenone was used as an internal standard. In HPLC method with UV/fluorescence detection (external standard method) LNG was monitored at 242 nm, while EED was detected with fluorescence detector at 310 nm (excitation 285 nm). The methods’ performances were fully validated by a determination of linearity, reproducibility, accuracy and sensitivity. Both methods were applied for determination of Uniformity of Dosage Units. The results obtained with both methods were highly comparable. However, the HPLC method with UV/ fluorescence detection has showed superior sensitivity for EED indicated by 83 times lower detection limit. HPLC method with UV/ fluorescence detection could be recommended as a method of choice for determination of ethinylestradiol, present at a very low dosage level in low-dose oral contraceptives, that also contain bigger amount of synthetic progestin.

Bakshi M., Singh S.

This write-up provides a review on the development of validated stability-indicating assay methods (SIAMs) for drug substances and products. The shortcomings of reported methods with respect to regulatory requirements are highlighted. A systematic approach for the development of stability-indicating methods is discussed. Critical issues related to development of SIAMs, such as separation of all degradation products, establishment of mass balance, stress testing of formulations, development of SIAMs for combination products, etc. are also addressed. The applicability of pharmacopoeial methods for the analysis of stability samples is discussed. The requirements of SIAMs for stability study of biotechnological substances and products are also touched upon.

Santoro M.I., Kassab N.M., Hasegawa M., Kedor-Hackmann É.R.

Görög S.

Publisher Summary This chapter presents the evaluation of impurities in hormonal steroids and mainly their synthetic analogues is dealt with. Other non-hormonal synthetic derivatives (e.g. quaternary ammonium derivatives) and pharmaceutically less important groups of natural steroids such as sterols and bile acids are only briefly mentioned. These are very advantageous features from the points of view of their determination by UV spectrophotometry in various matrices and the detectability in the course of the TLC or HPLC analysis but are of little importance when the analytical task is the direct determination of impurities/degradation products in bulk drugs and their formulations. Several spectrophotometric methods based on (colour) reactions are also available but the importance of their application to the determination of impurities in steroid drugs has been surpassed by modem chromatographic methods.

Miller R.B., Chen C.

A reversed-phase high-performance liquid chromatographic (HPLC) procedure using ultra-violet (UV) detection for the analysis of 17β-estradiol-3-phosphate in an ophthalmic solution is reported. The method is selective, accurate, and reproducible. The peak area versus 17β-estradiol-3-phosphate concentration is linear over the range of 50–150% of its label claim of 1.0 mg/mL, with a detection limit of 20 ng/mL. The mean absolute recovery of 17β-estradiol-3-phosphate using the described method is 99.8±0.6% (mean ±SD, n=10). A stress study with heat, acid, base and UV radiation indicates that the method is stability-indicating with no interference from excipients or degradation products.

Miller R.B., Delker G., Chen C., Sherwood C.H.

Abstract A validated reversed-phase high-performance liquid chromatographic (HPLC) procedure for the analysis of 17β-estradiol-3-phosphate is reported. In the development of this assay, several factors were evaluated including buffer ionic strength, mobile phase pH, ion-pairing concentration, organic composition, and column type. The described method is rapid and coupled with standard HPLC procedures leads to a selective, accurate, and reproducible assay. The peak area versus 17β-estradiol-3-phosphate concentration is linear over the range of 0.1–100 μg/mL, with a detection limit of 0.02 μg/mL.

Fernández N., García J.J., Diez M.J., Terán M.T., Sierra M.

A method for the determination of ethynyloestradiol in samples of rabbit plasma containing pentobarbital and heparin, the former used as an anaesthetic and the latter as an anticoagulant, has been developed. Quantification was carried out using a reversed-phase high-performance liquid chromatographic (HPLC) method in isocratic mode at room temperature, with electrochemical detection at an applied potential of +1 V vs. Ag/AgCl. Under these conditions, the retention time for ethynyloestradiol was ca. 2.9 min, the average recovery from plasma was 74.5%, and the limit of detection was 10 pg, corresponding to a plasma concentration of 50 pg/ml using 1 ml of plasma. Natural oestrogens, oestriol, oestradiol and oestrone showed peaks that did not interfere with ethynyloestradiol, and retention times of ca. 0.8, 2.4 and 3.4 min, respectively.

Qinjian Z., Zhensu L.

A procedure for the preparative resolution of ( E )- and ( Z )-norgestimate (17β-acetyloxy-13-ethyl-18,19-dinorpregn-4-en-20-yne 3-oxime) using f

Ahmed A.N., El-Gizawy S.M., Omar N.M.

A specific and sensitive analytical HPLC procedure was described for quantitative determination of ethinylestradiol and norethisterone acetate (Anovlar 1) and ethinylestradiol and norgestrel (Primovlar) in tablet formulation. These steroids were extracted from the tablets with methanol. The steroids were then determined with high performance liquid Chromatograph-Cyclobond 1 column using mobile phase phosphate buffer pH 7.0: methanol (60:40), flow rate 0.5 ml min−1 and the detection was effected spectrophotometrically at 280 nm, using variable wavelength UV detector. There was > 99.3% recovery from synthetic mixtures and the coefficient of variation was < 2.0% for the formulations investigated. The method is highly quantitative and reproducible.

Matlin S.A., Jiang L., Roshdy S., Zhou R.