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2004 © The Japan Society for Analytical Chemistry



Recent Advances in the Analysis of Steroid Hormones
and Related Drugs
Sándor GÖRÖG
Gedeon Richter Ltd., P.O.B. 27, H-1475 Budapest, Hungary

The development during the last 15 years and the state-of-the-art in the analysis of bulk steroid hormone drugs and
hormone-like structures and pharmaceutical formulations made thereof are summarized. Other steroids (sterols, bile
acids, cardiac glycosides, vitamins D) as well as biological-clinical aspects and pharmacokinetic and metabolic studies
are excluded from this review. The state-of-the-art is summarized based on comparisons of monographs in the latest
editions of the European Pharmacopoeia, United States Pharmacopoeia and the Japanese Pharmacopoeia. This is
followed by sections dealing with new developments in the methodology for the fields of spectroscopic and
spectrophotometric, chromatographic, electrophoretic and hyphenated techniques as well electroanalytical methods. The
review is terminated by two problem-oriented sections: examples on impurity and degradation profiling as well as
enantiomeric analysis.
(Received January 14, 2004; Accepted February 2, 2004)
1 Introduction
2 Steroid Hormone Drugs in Pharmacopoeias
2·1 Assay of bulk drug materials
2·2 Related impurities test of bulk drug materials
2·3 Assay of steroid hormone formulations
3 Spectroscopic and Spectrophotometric Methods
3·1 Ultraviolet-visible spectrophotometry
3·2 Fluorimetry
3·3 Infrared and Raman spectroscopy
3·4 Mass spectrometry
3·5 NMR spectroscopy
3·6 Chemiluminometry
3·7 Circular dichroism (CD) spectropolarimetry
4 Chromatographic and Hyphenated Methods
4·1 Planar chromatography
4·2 Gas chromatography (GC) and GC-MS




1 Introduction
The aim of this paper is to give an overview of the advances
during the last 15 years and of the state-of-the-art in the analysis
of steroid hormone drugs and related materials. Why just 15
years? My first book on steroid hormone drug analysis was
published in 19782 when this area was in transition state due to
the introduction and rapid spreading of new techniques, mainly
high-performance liquid chromatography (HPLC) and
immunoassay methods. This was followed by another book in
19833 dealing with wider areas (in addition to the analysis of
hormone drugs other types of steroids and biological-clinical
aspects). The third book, published in 19894 (just 15 years ago),
dealt mainly with pharmaceutical and industrial aspects. The
This paper is Part 56 in a series on “Analysis of Steroids”; for
Part 55 see Ref. 1.

4·3 Supercritical fluid chromatography (SFC)
4·4 High-performance liquid chromatography
5 Electrophoretic and Related Methods
5·1 Capillary electrophoresis (CE)
5·2 Micellar electrokinetic chromatography (MEKC)
5·3 Microemulsion electrokinetic chromatography
5·4 Capillary electrochromatography (CEC) and
6 Electroanalytical Methods
7 Selected Analytical Tasks
7·1 Impurity and degradation profiling of steroid
7·2 Enantiomeric analysis
8 References

development in steroid hormone drug analysis during the last 15
years can be characterized by the facts that HPLC undoubtedly
became the most important routinely used method and that the
importance of hyphenated chromatographic–spectroscopic
techniques is increasing. New techniques, mainly capillary
electrophoresis (CE) and related techniques, such as micellar (or
microemulsion) electrokinetic chromatography (MEKC,
MEEKC), capillary electrochromatography (CEC), also attract
wide interest in steroid analysis. Summarization of the results
achieved with these methods will be the subject of this review.
Only hormone drugs, their semi-synthetic analogues and
hormone-like structures are considered. Other groups of
steroids (sterols, bile acids, cardiac glycosides, vitamins D, etc.)
are not within the scope of this review. Pharmaceuticalindustrial aspects are discussed, such as assay and impurity
profiling of bulk drugs and drug formulations, estimating
pharmacokinetic and metabolic studies are also not within the
scope of this review. These aspects are dealt with in a book of

). even in those cases when the assay is carried out by HPLC methods. The outdated and labor-consuming method of TLC separation and UV spectrophotometry after spot elution is prescribed for the assay of meprednisone. Eur. This method is naturally non-specific: the overwhelming majority of the impurities are measured together with the main component. For the thin-layer chromatographic purity test in the majority of cases.7 with acetous perchloric acid. rather outdated methods2–4. but characteristic. Jp.or 1. since the European Pharmacopoeia does not contain monographs for formulations .or overestimation) if the chromophoric system or the color and/or the intensity of the TLC spot of the main component and the impurity are different.7 “Related steroids”7 or “Other steroids”. is restricted to a few cases. It is to be noted that UV spectrophotometry around 240 nm with limits for the specific absorbance is often part of the monographs as an identification test. both the TLC and HPLC tests express the impurities as the main component. this test is carried out in all cases by semi-quantitative TLC or quantitative (mainly but not exclusively reversed phase) HPLC methods. Jp. Eur. heating and visual inspection of the plate and the use of long-wavelength UV light (366 nm). IV. In spite of this limitation. prednisolone hemisuccinate and testosterone.8 As can be seen in Table 1.10 is not better than that of the spectrophotometric methods based on native absorbance measurements. spectra. Jp. iodine vapor (TLC–I2) and alkaline Blue Tetrazolium (TLC–tetrazolium). XIV).4-diene-3-oxosteroids. Eur.5 Spectroscopic studies are not discussed either. are also suitable for titrimetric determination.4diene-3-oxo group and at about 280 for estrogens with a phenoltype ring A with its weak. In the case of HPLC tests. in one instance (mestranol)7 the assay is based on a non-stoichiometric color reaction with a methanol–sulfuric acid reagent (545 nm). The wavelength of the UV detector is set most often at 254 nm. with two exceptions (conjugated and esterified estrogens. moreover. is prescribed by the European and United States Pharmacopoeias. Of the chromatographic methods other than HPLC. 20 pancuronium bromide6. Other titrimetric methods are restricted to the titration of ANALYTICAL SCIENCES MAY 2004. which is not the maximum of the spectrum of 4-ene-3-oxo. 2·3 Assay of steroid hormone formulations In this section the state-of-the-art in the field of determining the active ingredient content of steroid hormone formulations is based mainly on USP XXVI. The predominant method in the United States Pharmacopoeia is highly specific reversed phase (in some cases normal phase) HPLC. is UV spectrophotometry at about 240 nm for steroids with strongly absorbing 4-ene-3-oxo-or 1. nandrolone phenylpropionate. unless these are part of impurity profiling and degradation profiling studies. and Ph. These appear as “TLC 254 nm” in Table 1. much more important than the assay. (Ph. Although the overwhelming majority of steroid hormone drugs do not posses functional groups suitable for titration. The lactone group in oxandrolone.768 Makin et al.8 and stanozolol6. IV. In the Japanese Pharmacopoeia the HPLC and UV spectrophotometric assay methods are almost equally represented. the drugs and their impurities appear as dark spots in the chromatogram suitable for their semi-quantitative estimation. gas chromatography is only very seldom used for the assay of bulk steroid hormone drugs. such as a measurement around 380 nm after condensation of the unsaturated 3-oxo group with isoniazide. potassium dichromate/sulfuric acid (TLC–acid dichromate). It is interesting to note. As can be seen in Table 1.9. XIV do not contain tests for related impurities (27 and 10%. Jp. such as the extraction of free corticosteroids from the solution of their water soluble esters. Reagents containing sulfuric acid are most frequently used. or elution of TLC spots followed by UV spectrophotometric measurement or gas chromatography.mol. porous octadecylsilica support (3 – 10 µm) is used for the RP and porous silica support (5 – 10 µm) for the NP separations. In some cases the use of various visualizing reagents is necessary. VOL. “TLC–H2SO4” in Table 1 means spraying with this reagent. It is amazing that in many cases the USP XXVI and Ph.6 United States Pharmacopoeia 26th ed.7 2·2 Related impurities test of bulk drug materials The test for “Related substances”. Since most of the steroid hormone drugs strongly absorb UV light at this wavelength. 2·1 Assay of bulk drug materials Table 1 shows a brief summarization of the assay and related impurities tests in the monographs of bulk steroid hormones and hormone-like structures in European Pharmacopoeia 4th ed.8 is the most important test to characterize the quality of a bulk drug material. a UV detector is exclusively used at the wavelength of the maximum of the main component or at 254 nm. The proportion of HPLC tests for related impurities in bulk steroids in various pharmacopoeias is quite different: about 75% in Ph. IV). respectively). With a few exceptions. in some cases this method is still used. The use of other methods. the state-of-the-art of methodology and requirements in official drug analysis is well reflected by monographs concerning the latest revisions of the principal pharmacopoeias. where gas chromatography is used). sorbent layers are used that are impregnated with dyes strongly fluorescing when irradiated at 254 nm by a mercury lamp. This is attributable to the early period in the history of HPLC when the majority of instruments were equipped with only a mercury lamp. or an indirect measurement at 525 nm after a reaction with Tetrazolium Blue of corticosteroids with reducing side chain. 2 Steroid Hormone Drugs in Pharmacopoeias Pharmacopoeias are naturally rather conservative: new techniques appear in their monographs only if proved by many years of practice that the performance of these methods is superior to that of the currently used methods. and as a “Limit of ethynyl group” by the United States Pharmacopoeia. Other visualizing reagents are phosphomolybdic acid (TLC–phosph. Eur. (USP XXVI)7 and the 14th edition of the Japanese Pharmacopoeia (Ph. and difficult to explain that in some instances visible spectrophotometric methods.7 after opening it with sodium hydroxide and the –O–SO2–ONa group in sodium prasterone sulfate8 after Na+ → H+ ion exchange. p-toluenesulfonic acid. Ethinylsteroids can be titrated with sodium hydroxide after a reaction with silver nitrate and the liberation of nitric acid. 50% in USP XXVI and 10% in Ph. which can be source of serious errors (under.7 “Ordinary impurities”. especially if the latter is carried out by non-specific methods. XIV. vanillin/sulfuric acid (TLC–vanillin). The selectivity of these. the most frequently used assay method in Ph.6 also named “Chromatographic purity”. This method is used as a (non specific) assay method by Ph. In the overwhelming majority of cases.

4 USP XXVI Ph. VOL. esterified Estrone Estropipate Ethinylestradiol Ethynodiol diacetate Finasteride Fludrocortisone acetate Flumetasone pivalate Flunisolide Fluocinolone acetonide Fluocinonide Fluocortolone pivalate Fluorometholone Related impurity Ph. 4 — RP-HPLC 254 nm — — — VIS–tetrazolium RP-HPLC 254 nm — — RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm UV 238. Eur. XIV Ph. 20 Table 1 769 Assay and related impurities tests in European. — RP-HPLC 200 nm — RP-HPLC 210 nm RP-HPLC 215 nm UV 238 nm VIS–tetrazolium — — RP-HPLC 210 nm RP-HPLC 210 nm RP-HPLC 254 nm TLC 254 nm — — UV 239 nm VIS–tetrazolium — RP-HPLC 254 nm TLC–H2SO4 — — UV 238 nm RP-HPLC 254 nm — — TLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 238 nm — — UV 242 nm RP-HPLC 254 nm RP-HPLC 254 nm — RP-HPLC 254 nm TLC–tetrazolium — — RP-HPLC 243 nm — — — RP-HPLC 254 nm RP-HPLC 254 nm — — — RP-HPLC 254nm TLC 254 nm Continued .5 nm UV 241. Eur.Jp.5 nm RP-HPLC 254 nm — — — — RP-HPLC 254 nm — — — — RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm TLC 254 nm RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm — RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm — UV 235 nm — — NP-HPLC 254 nm — — — UV 238 nm NaOH UV 231 nm — UV 280 nm UV 281 nm GC — — — Titration ethinyl — — GC RP-HPLC 280 nm — RP-HPLC 205 nm — — RP-HPLC 230 nm RP-HPLC 280 nm — RP-HPLC 280 nm — UV 281 nm RP-HPLC 280 nm GC — GC — RP-HPLC 280 nm — RP-HPLC 213 nm — RP-HPLC 280 nm Titration ethinyl RP-HPLC 200 nm — — NP-HPLC 254 nm — — — RP-HPLC 280 nm RP-HPLC 230 nm — RP-HPLC 220 nm NP-HPLC 254 nm GC — — TLC–vanillin RP-HPLC 280 nm — NP-HPLC 280 nm — — TLC 254 nm — — — TLC–H2SO4 TLC–H2SO4 TLC–H2SO4 GC — GC — — — TLC–H2SO4 — RP-HPLC 213 nm — RP-HPLC 280 nm — Estrone–colorim.5 nm UV 238. USP and Japanese pharmacopoeias Assay Alclometasone dipropionate Amcinonide Beclomethasone dipropionate Betamethasone -acetate -benzoate -dipropionate -sodium phosphate -valerate Budesonide Chlormadinone acetete Clobetasol propionate Clobetasone butyrate Clocortolone pivalate Cortisone acetate Cyproterone acetate Danazol Desoxycort(icoster) one acetate -pivalate Desoximetasone Dexamethasone -acetate -sodium phosphate Diflorasone diacetate Drostanolone propionate Dydrogesterone Estradiol -benzoate -cypionate -valerate Estriol Estrogens. conjugated Estrogens.5 nm UV 240 nm — UV 240 nm UV 241 nm RP-HPLC 240 nm RP-HPLC 240 nm RP-HPLC 254 nm — RP-HPLC 254 nm — RP-HPLC 254 nm UV 239 nm RP-HPLC 254 nm RP-HPLC 254 nm UV 240 nm RP-HPLC 240nm — — USP XXVI TLC 254 nm — — TLC–H2SO4 RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm TLC–H2SO4 TLC 254 nm TLC 254 nm RP-HPLC 254 nm Betamethasone– extraction 239 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm — — RP-HPLC 240 nm — — UV 285 nm — — Ph.ANALYTICAL SCIENCES MAY 2004. Jp XIV — — TLC–tetrazolium TLC 254 nm — — TLC 254 nm TLC 254 nm TLC–tetrazolium — RP-HPLC 236nm RP-HPLC 240 nm — — RP-HPLC 240 nm — — — RP-HPLC 241 nm — — VIS–isoniazid — — TLC/elut 238 nm — UV 237 nm UV 282 nm RP-HPLC 254 nm — — — RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm — — — — UV 240 nm UV 285 nm VIS–tetrazolium — — — TLC–I2 RP-HPLC 254 nm — — — — — UV 238.

extraction RP-HPLC 254 nm 239 nm UV 240 nm VIS–tetrazolium — Hydrocortisone. — — — — Titration ethinyl — — Titration HClO4 Ion-exchange/ Titration NaOH RP-HPLC 243 nm — — UV 243.5 nm NP-HPLC 254 nm RP-HPLC 247 nm UV 243 nm NP-HPLC 254 nm RP-HPLC 254 nm — TLC 243 nm extr. — — TLC–I2 — TLC–H2SO4 — RP-HPLC 254nm TLC–H2SO4 — — RP-HPLC 244 nm NP-HPLC 210 nm TLC–phos. — TLC–H2SO4 RP-HPLC 254 nm RP-HPLC 254 nm — — — — RP-HPLC 280 nm — RP-HPLC 254 nm — RP-HPLC 265 nm — TLC 238 nm extr.5 nm -butyrate — -hydrogen UV 241. — — — — Titration ethinyl UV 240 nm Titration ethinyl TLC–H2SO4 Titration ethinyl UV 240 nm Titration ethinyl RP-HPLC 254 nm — — Titration ethinyl — — Titration HClO4 — UV 240 nm–HCl RP-HPLC 244nm UV 241 nm Titration lactone TLC 315 nm extr.mol. TLC 254 nm TLC–H2SO4 — — — — TLC–NaNO2+BiI3 — TLC–H2SO4 RP-HPLC 243 nm — — RP-HPLC 254 nm TLC 254. — — RP-HPLC 200 nm — — TLC–p-toluenesulfonic acid UV 279 nm TLC–H2SO4 VIS–H2SO4 — UV 242 nm — — UV 242 nm NP-HPLC 254 nm UV 243 nm — — — — RP-HPLC 241 nm UV 241 nm RP-HPLC 254 nm — RP-HPLC 254 nm — — TLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm — — — — — TLC–H2SO4 — — — — TLC–H2SO4 TLC 254 nm — TLC 254 nm RP-HPLC 254 nm TLC–tetrazolium — — — — TLC 254 nm RP-HPLC 254 nm TLC 254 nm — — — RP-HPLC 254 nm TLC 254 nm — — RP-HPLC 240 nm — — NP-HPLC 238 nm — — TLC 239 nm extr. — UV 243 nm — — UV 241 nm UV 247 nm — — — TLC–phos. VOL.366 nm TLC–tetrazolium RP-HPLC 254 nm NP-HPLC 254 nm TLC 254 nm — — — RP-HPLC 254 nm — — RP-HPLC 254 nm Prednisolone — Continued . RP-HPLC 254nm extraction 239 nm — TLC 254 nm NP-HPLC 254 nm — — RP-HPLC 254 nm RP-HPLC 254 nm — UV 240 nm — — — — — NP-HPLC 254 nm — — TLC 254 nm — — — TLC–H2SO4 NP-HPLC 254 nm — — RP-HPLC 254 nm — UV 241 nm — RP-HPLC 254 nm — — — TLC–phos.366 nm — — — RP-HPLC 239 nm — — UV 239 nm — — NP-HPLC 254 nm NP-HPLC 254 nm RP-HPLC 254 nm NP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm RP-HPLC 254 nm UV 241 nm — NP-HPLC 254 nm — RP-HPLC 254 nm TLC/elut. 20 Fluoxymesterone — Flurandrenolide — Fluticasone RP-HPLC 239 nm propionate — Halcinonide Hydrocortisone UV 241.5 nm (hemi)succinate -sodium — phosphate -sodium — succinate -for injection — -succinate — -valerate — Hydroxyprogesterone — caproate Isoflupredone — acetate Levonorgestrel Titration ethinyl Lynestrenol Titration ethinyl Medroxyprogester. TLC–phos.5 nm -acetate UV 241.mol.mol.UV 241 nm one acetate Megestrol acetate UV 287 nm Mepitiostane — Meprednisone — Mesterolone RP-HPLC 254 nm Mestranol Metenolone acetate Metenolone enanthate Methylprednisolone -acetate -hydrogen succinate Methyltestosterone Mibolerone Mometasone furoate Nandrolone decanoate Nandrolone phenylpropionate Norethisterone (norethindrone) -acetate Norethynodrel Norgestimate Norgestrel Oxandrolone Oxymetholone Pancuronium bromide Prasterone sodium sulfate Prednicarbate Prednisolone -acetate -hemisuccinate -pivalate -sodium Titration ethinyl — — UV 243 nm UV 243 nm UV 243 nm UV 241 nm — UV 249 nm RP-HPLC 254 nm NP-HPLC 254 nm — RP-HPLC 254 nm TLC 254 nm RP-HPLC 240 nm — — TLC 254.mol. 239 nm — NP-HPLC 254 nm TLC 254 nm RP-HPLC 254 nm TLC–tetrazolium RP-HPLC 254 nm TLC–tetrazolium RP-HPLC 254 nm — Enzime.770 ANALYTICAL SCIENCES MAY 2004.

gel. heptane. mainly with chloroform or diluted with hexane. dexamethasone acetate injectable suspension. norethindrone and ethinylestradiol tablets. etc. hydrocortisone cream. followed by extraction with polar solvents or solvent mixtures. TLC separation. estriol tablets. lotion. medroxyprogesterone acetate injectable suspension. Direct extraction with methanol. XIV contains only a few (the formulations in Pharm. flunisolide nasal solution. prednisone oral solution. spironolactone tablets. ointment. fluorometholone ophthalmic suspension. injectable suspension and vaginal suppositories. Oily injections are either simply diluted. cortisone acetate injectable suspension. estradiol benzoate injectable suspension. fluocinolone acetonide cream. ointment and topical solution. gel and topical solution. 2-propanol or the mobile phase of the RP-HPLC system. dexamethasone injection. or extraction of the aqueous phase with chloroform. esterified and conjugated estrogen tablets. rectal suspension. fluocinonide cream. ophthalmic ointment and ophthalmic solution. acetonitrile or the mobile phase of the RP-HPLC system. desoxymetasone cream and ointment. etc. ethyl acetate etc. GC VIS–isoniazid — RP-HPLC 254 nm -propionate UV 241 nm Trenbolone acetate — UV 241 nm VIS–isoniazid RP-HPLC 344 nm — TLC 254 nm — TLC–H2SO4 RP-HPLC 240 nm TLC 254 nm — Triamcinolone -acetonide -diacetate -hexacetonide RP-HPLC 254 nm RP-HPLC 254nm RP-HPLC 254 nm RP-HPLC 254nm RP-HPLC 254 nm — RP-HPLC 254 nm — RP-HPLC 238 nm RP-HPLC 254 nm — RP-HPLC 254 nm UV 238 nm UV 238. fluoxymesterone tablets. betamethasone benzoate gel. either at 254 nm or at the maximum of the UV spectra of the active ingredients. Jp. mometasone furoate cream. while in addition to these. triamcinolone tablets. ointment. acetonitrile. syrup and injectable suspension and triamcinolone hexacetonide injectable suspension. acetonitrile. betamethasone sodium phosphate and acetate injectable suspension. ophthalmic suspension. spironolactone and hydrochlorothiazide tablets. progesterone injection. hydrocortisone tablets. clobetasol propionate cream. triamcinolone diacetate oral suspension.ANALYTICAL SCIENCES MAY 2004. estropipate tablets and vaginal cream. megestrol acetate oral suspension and tablets. ointment. fludrocortisone acetate tablets. More common is direct extraction mainly with methanol. oxandrolone tablets and testosterone cypionate injection. — RP-HPLC 344 nm — — RP-HPLC 254 nm TLC 254 nm — — RP-HPLC 254 nm — estradiol cypionate and valerate injection. halcinonide cream and ointment. rimexolone ophthalmic suspension. Normal-phase HPLC is used for the assay of cortisone acetate tablets. betamethasone valerate cream. prednisolone tablets. is also used for ointments. ointment and topical solution. methylprednisolone acetate injectable suspension. ointment. drostanolone propionate ointment. This method is used among others for the assay of aclometasone dipropionate and amcinonide cream and ointment. injectable suspension and tablets. etc. oral solution elixir and tablets. extraction followed by Tetrazolium . extract. topical aerosol. triamcinolone acetonide cream. ioctane followed by extraction of the apolar phase with mixtures of water and methanol. chloroform. danazol capsules. norethindrone and mestranol tablets. prednisolone tablets and prednisolone tebutate injectable suspension. finasteride tablets. lotion and injectable suspension. levonorgestrel and ethinylestradiol tablets. is the most widely used sample-preparation method for creams. The predominant method for the assay is reversed phase HPLC with UV detection. Of the other chromatographic methods. dexamethasone sodium phosphate cream. VIS–tetrazolium 771 phosphate -sodium succinate -succinate -tebutate Prednisone Progesterone Rimexolone Spironolactone — — UV 238 nm UV 241 nm — UV 238 nm Stanozolol Testolactone Titration HClO4 — — RP-HPLC 242 nm — NP-HPLC 254 nm — — RP-HPLC 254 nm — RP-HPLC 254 nm RP-HPLC 254 nm UV 241 nm RP-HPLC 241 nm RP-HPLC 242 nm — — RP-HPLC RP-HPLC 254 nm UV 238 nm 254/283 nm Titration HClO4 — TLC–H2SO4 — — VIS–isoniazid Testosterone -cypionate -enantate UV 241 nm — UV 241 nm TLC 239 nm extr. A great variety of sample-preparation methods are used to assay the formulations. hydrocortisone valerate cream and ointment. prednisolone oral suspension and syrup. desoxycorticosterone pivalate injectable suspension. or extracted with chloroform. mibolerone oral solution. extraction 241 nm — — — TLC 254 nm — — NP-HPLC 254 nm — — TLC 254 nm RP-HPLC 242 nm — TLC–H2SO4 TLC–H2SO4 TLC–H2SO4 — TLC 254 nm + — acid dichromate — — — — — TLC–p-toluene sulfonic acid — TLC 254 nm TLC–phos. lotion. 20 enzime.mol. medroxyprogesterone acetate tablets. flurandrenolide cream.) are either disintegrated with water. Aqueous suspensions are homogenized by simple dilution with methanol. estrone injectable suspension. diflorasone cream. lotion and topical aerosol. ointment and lotion. nandrolone decanoate injection. ointment and topical solution. Solid dosage forms (tablets. methylprednisolone tablets. gas chromatography is used for the assay of drostanolone propionate injection. betamethasone dipropionate cream. Jp. extraction with hexane.5 nm — UV 238 nm — — UV 241 nm — and Pharm. betamethasone cream and tablets. prednisolone acetate injectable suspension and ophthalmic suspension. injection. dydrogesterone tablets. hydrocortisone acetate cream. fluorometholone cream. hydrocortisone butyrate cream. XIV are indicated by cursive letters). ointment and lotion. etc. isofluprednone acetate injectable suspension.. betamethasone sodium phosphate injection. norgestrel and ethinylestradiol tablets. Aqueous solutions are usually diluted with water or the mobile phase of the RP-HPLC method. estradiol tablets and vaginal cream. ethynodiol diacetate and ethinylestradiol tablets. followed by dilution with methanol. VOL. tape. ethynodiol diacetate and mestranol tablets.

Derivative UV spectrophotometry10 was also successfully applied to the analysis of steroid hormone formulations. In simple cases improved extraction procedure such as solid phase extraction in the case of the determination of hydrocortisone in ointments12 is sufficient. testosterone enanthate injection and testosterone propionate injection) or 4-aminoantipyrine (flumethasone pivalate cream) measure the less vulnerable part of the molecules. dydrogesterone tablets.6 nm with 277 nm as the reference wavelength.32 An interesting feature of another method for the assay of spironolactone and hydrochlorothiazide. naphazoline and dexamethasone. It is amazing that an extremely outdated and labor-extensive method is used by USP XXVI for the assay of estrone oily injection. norgestrel tablets.10 For example. such as the principal component regression method. A UV spectrophotometry is used in some other cases as well in the content uniformity and dissolution tests of tablet formulations.19 The accuracy and precision of the determination of even as low a quantity as 2 mg/vial dexamethasone in the presence of 250 and 10 mg of vitamins B6 and B12. Partial least-squares regression analysis was applied to other diuretic preparations.19 cyproterone acetate and estradiol valerate30 in pharmaceutical formulations. VOL. Finally the weight of estrone is measured! 3 Spectroscopic and Spectrophotometric Methods 3·1 Ultraviolet-visible spectrophotometry As shown in the previous sections.26 as well as ethinylestradiol and gestodene. such as spironolactone–althiazide15 and spironolactone–chlorthalidone16 formulations. Examples for this are the determination of spironolactone and hydrochlorothiazide. testolactone tablets. reactions based on the condensation of the unsaturated 3-oxo group with isoniazid (clocortolone pivalate cream. while column chromatographic separation followed by Tetrazolium Blue colorimetry or iron-phenol colorimetry is used for the assay of dexamethasone gel and estradiol pellets and injectable suspension. hydrocortisone injectable suspension and hydrocortisone acetate ophthalmic ointment and suspension. 50 ml of benzene (!) and 95 ml of chloroform). making use of a rapidscanning diode-array spectrophotometer. The validation of the determination of the latter by interlaboratory study preceded by recrystallization and checking the purity of the test sample by phase solubility analysis. The procedure includes three extraction steps (consuming 50 ml of hexane. stanozolol tablets and testosterone injectable suspension. The reactions of the phenol-type ring A based on diazo-coupling (estradiol benzoate injection) or on the nonstoichiometric reaction with the sulfuric acid–methanol reagent (ethinylestradiol tablets) are not stability-indicating either. In simple cases (single-component formulations) only the spectral background caused by the excipients should be eliminated.20 Other 3-component formulations containing low quantities of dexamethasone resolved by partial least-squares regression analysis and other chemometric methods were and Polymyxin B. prednisolone sodium phosphate injection and ophthalmic solution (after enzymatic hydrolysis and extraction). dexamethasone topical aerosol.11 Problems to be solved for the application of this method to the assay of drug formulations is the elimination of matrix effects and simultaneous ANALYTICAL SCIENCES MAY 2004. Most of their degradation products do not react with the alkaline Tetrazolium Blue (desoxycorticosterone acetate injection and pellets. The above-discussed chromatographic methods are selective and stability indicating. The same cannot be said for the nonchromatographic methods. formation of hydrazone derivative with trimethylacethydrazide ammonium chloride and its decomposition. based on ratio spectrum derivative spectroscopy.22 In the latter case.14 The performance of the method can be improved by multiwavelength measurements and chemometric methods. respectively. oxymetholone tablets.27 A zero-crossing second-derivative method was used for the determination of fluorometholone and tetrahydrozoline hydrochloride28 as well as estradiol and medroxyprogesterone acetate29 in formulations. the use of artificial neural network analysis was necessary for the determination of dexamethasone due to the non-linearity caused by interactions among the components. nystatin and oxytetracycline.9. the release of hydrocortisone acetate from microcapsules in aqueous suspension was monitored at 247.13 The classical dual-wavelength spectrophotometry was successfully used for the simultaneous determination of spironolactone and hydrochlorothiazide with their overlapping spectra in a tablet formulation. For this purpose first-derivative spectra are usually sufficient. dexamethasone21 chlorpheniramine.772 Blue colorimetry is the assay method for betamethasone syrup and methylprednisolone acetate cream.2–4. in spite of its poor selectivity UV spectrophotometry is widely used for the identification and assay of bulk steroid drugs. nandrolone phenylpropionate injection. Another possibility to improve the selectivity of the method is the use of “ratio spectrum (zerocross) derivative spectroscopic” method. in an injection formulation were good. reducing side chains at position 17. 20 determination of the active ingredients of two. injectable suspension) or phenylhydrazine–sulfuric acid reagents (hydrocortisone sodium phosphate injection and prednisolone cream). progesterone in intrauterine contraceptive system. trimethoprim. hydrocortisone caproate injection. One example is the mixture of hydrocortisone. methyltestosterone tablets and capsules. two evaporations to dryness. nystatin and oxytetracycline. is that it is adopted to . Dual-wavelength measurements can also give satisfactory results. The assay is carried out either using a reference standard material or by determining and describing the value of the specific absorbance. norethindrone tablets. The much more delicate problem of the assay of and the low-dosed levonorgestrel–ethinylestradiol17 gestodene–ethinylestradiol18 oral contraceptive tablets was also solved by the partial least-squares regression analysis. Even three-component drug formulations were successfully analyzed by this and other chemometric methods. The selectivity of some colorimetric methods also used for the assay of steroid hormone formulations is not better either. The situation is somewhat better with the color reactions of corticosteroids with sensitive. UV spectrophotometric assay based on natural absorption is carried out for dexamethasone sodium phosphate inhalation aerosol. HPLC and differential scanning calorimetry was described on the example of triamcinolone. An example for this is the determination of triamcinolone acetonide in an ointment.or multicomponent formulations. respectively. The performance of various multivariate chemometric and derivative spectrophotometric methods is compared for examples of formulations containing hydrocortisone and ZnBacitracin31 as well as dexamethasone and polymyxin B.14 hydrocortisone. For example.23 The same technique using the “zero-crossing” method was found to be suitable for the simultaneous determination of the components of dual-component drug formulations such as creams and tablets containing triamcinolone acetonide and terbinafine hydrochloride24 and oral contraceptives containing ethinylestradiol and levonorgestrel25.

condensation with isoniazid and phenylhydrazine. However.46 Near-infrared reflectance spectroscopy has become a versatile tool in drug analysis and in-process control in drug production. among others finasteride in their solid formulations. norethandrolone. methylboldenone.48 3·5 NMR spectroscopy One of the relatively rare direct applications of NMR spectroscopy in pharmaceutical steroid analysis is its use for the rapid screening and compositional analysis of steroid cocktails and veterinary drug formulations illegally used to livestock.34 Kinetic variants of the classical Blue Tetrazolium and phenylhydrazine35 and isoniazid36 reactions and their application to the analysis of corticosteroids in various pharmaceuticals are also worth mentioning.4. Many other reactions were also described. As an example. as seen in the sections dealing with pharmacopoeias. fluoxymesterone. especially Fourier-transform IR (FTIR) is one of the most important methods. two are mentioned here. redox reaction with tetrazolium reagents) are still in use in some instances. clobestol acetate. but also for discrimination between the two polymorphic forms Further analytical applications of NMR of the latter.9 have almost completely lost their importance in modern pharmaceutical analysis. differential scanning calorimetry (DSC). testosterone and its cypionate and decanoate and other steroids were identified directly or after HPLC fractionation with the aid of a 1H-NMR database. solid state 13CNMR is suitable not only for detecting low-dose active ingredients (among others prednisolone) in solid dosage forms. highly specific and sensitive FIA method for the determination of fluticasone propionate is based on splitting with sodium hydroxide of its –CO–S–CH2F side chain to form fluoromethylthiol which is further reacted with glycine and o-phthalaldehyde. as well as the content of various drugs. methanol–sulfuric acid is still an important spray reagent in the thin-layer chromatographic analysis of steroid drugs.45 TG/FT-IR studies supported by X-ray diffraction and DSC revealed the mechanism of the decomposition of prednisolone hemisuccinate in a freeze-dried formulation. only one direct application is mentioned. mass spectrometry is applied in pharmaceutical steroid analysis associated with various chromatographic techniques. e. The early literature of the applications to steroid hormone analysis was reviewed.54 3·7 Circular dichroism (CD) spectropolarimetry The main application field of CD spectroscopy is structural analysis but this technique can be successfully used also for analytical purposes.g.47 3·4 Mass spectrometry In the majority of cases. The sensitising effect of corticosteroids on the chemoluminogenic reaction between sulfite and cerium(IV) was exploited for their flow-injection (FIA) determination in pharmaceutical formulations. 3·2 Fluorimetry The native fluorescence of estrogens enables their direct fluorimetric determination.51 Similar studies related to chiral interactions are shown in the last section of this review. VOL. Examples for these will be presented in some of the subsequent sections.2–4. In addition to analytical applications. the transferred NOESY study of the interactions between various steroids and calix[8]arene-based stationary phases is mentioned.37 However.38 3·3 Infrared and Raman spectroscopy Infrared (IR) spectroscopy is the most generally used method for the identification of bulk steroid drugs in modern pharmacopoeias.ANALYTICAL SCIENCES MAY 2004. 20 flow-injection analysis. IR spectroscopy. Here.33 The importance of spectrophotometric/colorimetric methods based on chemical reactions has dramatically decreased in the last years both in the analysis of bulk steroid drugs and formulations made thereof. Measurements based on fluorescence induced by nonstoichiometric reactions with various strongly acidic reagents3. there are also a few direct applications in pharmaceutical analysis. Stanozolol. The strongly fluorescent isoindole derivative is formed in the reaction coil of the FIA apparatus.53 A luminol chemiluminescence determination for the same purpose is also described.39 One of the most characteristic examples is spironolactone.49 When using interrupted decoupling. Cross sections of the device containing a multi-polymer-layer system were probed for the distribution of the active ingredient. This is the case with a paper where Friedel–Crafts acetylation of steroids followed by taking the UV spectrum enables interesting discrimination to be made between various isomeric derivatives. Conventional IR spectrometry using fused KBr discs is not suitable for their differentiation due to the easy transformation of the polymorphs under pressure. FT-IR studies combined with all of the above mentioned solid-phase techniques revealed the existence of three polymorphs of fluocinolone acetonide43 as well as two polymorphs and a hemihydrate of flunisolide. On the basis of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and Fourier transform Raman spectroscopic investigations supported by DSC and TGA measurements40 four different polymorphic forms41 and five solvates42 were identified and characterized.55 Of the recent applications. the main application field of this is their sensitive HPLC detection. It was found to be suitable to solve as delicate a problem as the simultaneous determination of norethisterone and ethinylestradiol in oral contraceptive tablets.10 Further research in this field can be considered to be unnecessary: publications of this kind published in the last 15 years are not referred to in this review unless these are particularly interesting. the determination without a preliminary separation of estradiol valerate in an injection formulation in the presence of hydroxyprogesterone caproate (Ex: 295 nm/Em: 308 nm).44 The DRIFTS method is also suitable for the rapid verification 773 of identity. where in the early literature several polymorphs and solvates were described. Time-of-flight secondary ion mass spectrometry was successfully applied for the characterization and imaging of a controlled-release drug delivery system containing prednisolone sodium metasulfobenzoate. An interesting.9. The profound difference between the ellipticities of ethisterone and its 5-ene isomer .50 spectroscopy are discussed in the section dealing with HPLC.g. NMR spectroscopy is a good tool for studying the mechanism of interactions taking place between the analyte and stationary phase.6–8 In addition to this. together with X-ray powder diffractometry.52. As can be seen in Table 1 and in the section “Assay of steroid hormone formulations” some of the classical reactions (e. 3·6 Chemiluminometry Although the main application field of chemiluminescence in steroid analysis is chemiluminescence immunoassay in biological-clinical analysis. thermogravimetry (TGA) and thermomicroscopy to characterize the solvates and polymorphic modifications frequently occurring among steroid hormone drugs.

56 applicability of the CD method can be enhanced by the use of preliminary chemical reactions... comparison of the impurity profile with those obtained by TLC. phosphomolybdic acid or sulfuric acid).774 enables their selective determination (ethisterone at 348 nm at its negative maximum of the CD spectrum) and the isomer at 296 nm at the positive maximum of the CD curve. The densitograms were scanned in the reflectance mode at wavelengths which correspond to the isobestic point of the two components: 228 nm for betamethasone–clotrimazole cream. HPLC and GC).70 dexamethasone sodium phosphate–neomycin sulfate solution (246 nm). This method was found to be suitable for the determination to as low a concentration as 0. selectivity) of TLC and HPTLC were compared using steroids as the model compounds.83 programmed multiple development TLC with a new modification of the horizontal sandwich chamber84 and ultra-thin-layer chromatography (UTLC) where the granular adsorbents are replaced by a monolithic structure based on a silica-gel matrix has been demonstrated by using. e.57 4 Chromatographic and Hyphenated Methods 4·1 Planar chromatography In contrast to the pharmacopoeias where TLC is used only as a semi-quantitative limit test for purity check of bulk steroid hormone drugs. VOL. Some of the important parameters (retention behavior.80 OPLC was successfully used in in-process-monitoring81 and cleaning validation of equipment82 in the course of the production of bulk steroids and steroid formulations. 20 hydrocortisone.60 The determination of the six components of conjugated estrogens in raw materials and tablet formulations was carried out after hydrolysis. As a result of these factors.g. The advantages of this technique are clear: due mainly to the smaller particle size (about 4 µm) and narrow particle-size distribution of the stationary phases. shorter running distance and shorter running time. respectively).74 Reversed-phase (ion pair) TLC74 and HPTLC75 systems were used for the assay of corticosteroid sodium phosphate salts in parentheral preparations and ear drops75 and various steroids. The reasons for this are their low volatility and (especially in the case of corticosteroids) thermal instability.61 If carefully validated.79 norethisterone drug substance and tablet (sulfuric acid). derivatized reversed-phase systems for steroid separations and with the elucidation of the separation mechanism.72 The basis for the assay of ANALYTICAL SCIENCES MAY 2004. The popularity of high-performance thin-layer chromatography (HPTLC) is continuously increasing: in the majority of papers published in the last 15 years the use of this method is reported. GC-MS is an ideal tool for screening purposes.88 and impregnation of the silica gel TLC plate with dcamphorsulfonic acid/copper(II) acetate for the separation of the diastereomeric 21-ester derivatives of prednisolone formed with racemic tetrahydrophthalic acid. separation efficiency.02% of levonorgestrel acetate impurity in norgestimate. Examples for the application of OPLC to steroid hormone drugs are the purity test of norgestrel and levonorgestrel (visualization by fluorescence quenching at 254 nm.86 A tandem TLC-HPLC method is described for screening cosmetic products for the presence of undeclared synthetic corticosteroids.89 Some papers deal with the possibilities of applying only seldom-used stationary phases e.69 dexamethasone and xylometazoline in nasal drops (240 nm).90. Overpressured thin-layer chromatography (OPLC) is a variant of HPTLC in which the sorbent layer is covered with a pressurized elastic plate thus eliminating the vapor phase. the The spectropolarimeter could act as a CD-detector..64 In the majority of cases.71 prednisolone acetate in pharmaceutical formulations (243 nm. The usefulness of further innovations. followed by TLC separation and detection at 280 nm. extraction. Due to the forced flow of the eluent delivered by an automatic pump its migration velocity is constant and the migration distance can be increased up to 18 cm thus enabling excellent separations and improved quantifiability to be achieved.65 betamethasone tablets (245 nm). In the case of sufficiently stable steroids it is a good method of choice for the assay of among others methyltestosterone (after dimethylethyl. gas chromatography does not play a very important role in the analysis of steroid hormone drugs. The principles and practice of validation were described taking danazol62 and estradiol benzoate63 as examples (scanning at 252 and 229 nm.and R(–)-budenoside on cellulose-coated HPTLC plates. such as temperaturecontrolled programmed TLC. among others. Typical papers describing the assay of formulations are e. and as seen in Table 1. the separation of steroid mixtures.77 allylestrenol drug substance and tablet (sulfuric acid).78 nandrolone (sulfuric acid.67 cyproterone acetate–ethinylestradiol tablets (284 nm)68 or ethinylestradiol (220 nm) and cyproterone acetate (284 nm). When the simultaneous determination was carried out by HPLC.g. (see section “Related impurities test of bulk drug materials”).66 mometasone furoate topical preparations (260 nm). A difference CD method was developed for the determination of 4-ene-3-oxo steroids based on oxime formation making use of the large difference between the ellipticities before and after oximation. the separation efficiency increases. hydrocortisone acetate and clioquinol containing ointment and cream preparations was fluorescence quenching at 254 nm.73 In the case of estradiol tablets (284 nm). in the literature of the last 15 years almost exclusively quantitative densitometric methods are described for the purity check and also for the assay of steroid hormone formulations. the quantitative TLC densitometric purity test is equivalent to the HPLC purity test.91 4·2 Gas chromatography (GC) and GC-MS As already mentioned earlier in this review. enabling well-separated and wellquantifiable spots to be achieved with smaller plates.85 An interesting study dealing with the reasons for and the elimination of the irreversible adsorption of some steroids during multiple development TLC merits mentioning.58 247 nm for betamethasone–clioquinol cream59 and 233 nm for betamethasone–miconazole nitrate cream.or and cyproterone acetate– dimethylisopropylsilylation)92 ethinylestradiol93 tablets.76 respectively. Conventional silica gel 60 F254 plates were used. As many as . silica gel 60 F254 plates were used for the assay of steroid preparations. Florisil. OPLC is eminently suitable for the separation of impurities and degradants in bulk drugs and their formulations. Some of the investigated formulations are as follows (the wavelength of the densitometric scan in parentheses): finasteride tablets (228 nm). the HPTLC method was suitable not only for the assay but also for the detection of degradants formed under stress conditions. the determination of betamethasone valerate in various creams together with other active ingredients.87 Chiral selectors improved the separation of diastereomeric steroid pairs. the addition of β-cyclodextrin to the mobile phase in the case of the separation of R(+). stepwise gradient).g.

152 Ultrafast analysis is also achievable by decreasing the column length with simultaneous optimization of the flow rate and gradient profile (ballistic gradients).148 estradiol and its 3acetate from intravaginal rings149 and triamcinolone acetonide from topical dosage forms.113 dexamethasone acetate–prednisolone tablet.143 identification of the products of biotechnological transformations.144 HPLC-UV was used as the in-process-control method to follow this reaction at the industrial scale.135 HPLC is eminently suitable for screening purposes.111 (St). e.99 Examples for the use of chiral SFC will be shown in the section “Enantiomeric separations”. In addition to the above discussed use of HPLC for the analytical investigation of bulk steroids and steroid formulations it is a useful tool also in the hands of pharmaceutical technologists to investigate drug release from various formulations and devices for controlled drug delivery. where the assay and purity test for several bulk drugs is presented in Table 1 followed by listing several formulations assayed by HPLC. its acetate and other corticosteroids in ointments. an increased carrier gas flow rate and a decreased temperature.153 Temperature-responsive stationary phases showing hydrophilic properties at lower while hydrophobic properties at higher temperatures were prepared by covalently binding poly(Nisopropylacrylamide) to aminopropylsilica.107. Allylestrenol–α-tocopherol tablets.154–156 Excellent separation of the epimers of estradiol was achieved by modifying the surface of silica gel with the covalent binding of cholesterol.142 Capillary HPLC-MALDI-TOFMS after post-column derivatization with 2.108 sodium–cyanocobalamine tablet.139 Especially useful is the coupling of HPLC with mass spectrometry: a HPLC/time-of-flight MS method was developed for screening anabolic steroids in illegal cocktails.129 triamcinolone acetonide dermatological patches. 20 134 underivatized drugs (among them 10 steroids) were included in a study to screen ethnic patent medicines available in health-food stores and ethnic markets.138 NP.157 Molecularly imprinted artificial receptors as .119 hydrocortisone–oxytetracycline–nystatin in pharmaceutical preparations.118 fluocortolone pivalate and hexanoate suppositories.122 hydrocortisone sodium phosphate gel (St).100 medroxyprogesterone acetate101 and cyproterone acetate101 from tablet matrices.134 and for the photodegradation kinetic study of danazol.151 among others ingredients of a topical cream containing triamcinolone acetonide and preservatives.125 prednisolone acetate–tetrahydrozoline hydrochloride– ofloxacin ophthalmic preparations.123 nestorone implants (St).102 beclomethasone dipropionate inhalers103 and other formulations (St).117 finasteride tablet (St).116 ethinylestradiol–norgestrel tablet. probably due to their vulnerable 17-side chain.132 spironolactone-β-cyclodextrin solutions.4dinitrophenylhydrazine was used for the screening of combinatorial libraries with 7 corticosteroids as model HPLC-MS was also used for the rapid compounds.109 cyproterone acetate tablet dexamethasone–xylometazoline nasal drops.128 triamcinolone. 4·3 Supercritical fluid chromatography (SFC) Although the problem of thermal instability in GC can be solved by using supercritical fluid chromatography. Examples are the release of prednisolone from chitosan gel beads147 or from chitosan-gelatin sponges. methyl. mainly C18 columns with unbuffered binary or ternary mixtures of water with methanol.and propylparaben topical cream (St). It is to be noted that two of the ten (dexamethasone and prednisolone) could not be detected by GC-chemical ionization MS.141 HPLC-MS is the most suitable method for the rapid screening of highthroughput drug mixtures.136 corticosteroids in topical pharmaceuticals using diode-array UV detector.105.120 hydrocortisone acetate and hemisuccinate–lidocaine in pharmaceutical preparations. in the majority of cases reversed-phase HPLC is used. Supercritical fluid extraction can be a useful alternative to classical extractions as demonstrated on the examples of megestrol acetate.145 On-line HPLC-NMR—although only few data are available for its use in pharmaceutical steroid analysis—is certainly the method of the future even in this field. acetonitrile or tetrahydrufuran.98.106 betamethasone and dexamethasone (delicate betamethasone–diclofenac separation problem!).95 Another screening study using GC-MS aimed at detecting anabolic steroids96 and prohormones97 in nutritional supplements.and propylparaben topical cream (St).150 As mentioned earlier. Some further examples are briefly summarized here. Several steroids were separated by SFC with lightscattering detection.133 aqueous pipecuronium bromide.94 One possibility to enhance the applicability of GC and GC-MS to some of the thermally labile compounds is “supersonic GC-MS” using shorter capillaries with lower film thickness. This is a promising tool for the temperature-controlled separation of steroids. RPHPLC for anabolic steroids.g. VOL.114 estradiol– levonorgestrel transdermal drug delivery formulation (St). For example. its acetate and acetonide and several other corticosteroids (St).140 while for their determination in oily formulations RP-HPLC separation followed by off-line GC-MS analysis was used. this technique seems to have attracted little interest in steroid analysis. The model for this study was the hydroxylation of progesterone to 9α-hydroxyprogesterone.130 triamcinolone acetonide.and RP-HPLC for corticosteroids.126 prednisolone sodium phosphate implantable infusion pump (St).ANALYTICAL SCIENCES MAY 2004. Some innovations regarding stationary phases are as follows.121 hydrocortisone acetate–methyl.110 dexamethasone–retinol palmitate ointment. using this technique estradiol and norethisterone acetate were detected in a galenic emulsion.99 The sensitivity of the laser light scattering detector is similar to that of the UV detector with the advantage that UV-inactive steroids can also be detected with the same sensitivity. This enables several steroids (among them corticosterone with a vulnerable side chain) to be determined.127 progesterone in 775 micellar systems and other formulations.124 pancuronium bromide injection (St).131 Carefully validated stability-indicating HPLC methods were used for the determination of impurities and degradants in bulk budesonide. 4·4 High-performance liquid chromatography (HPLC) and HPLC-MS HPLC as the most important and most generally used method in pharmaceutical steroid analysis is described and characterized in the chapter “Steroid hormone drugs in pharmacopoeias”.104 betamethasone.114 ethinylestradiol– levonorgestrel and ethinylestradiol–gestodene tablets.146 Further examples for the use of HPLC coupled with spectroscopic methods are presented in the section “Impurity and degradation profiling of steroid drugs”. Monolithic columns were found useful tools for the ultrafast separations of steroids. taken from the literature where many “pharmacopoeial-like” reversed-phase HPLC assays for formulations are described (the abbreviation “St” in parentheses relate to stability assays).115 estradiol valerate–medroxyprogesterone acetate tablet.137 toxicological analysis of steroids and other compounds with diode-array UV detector.112 dexamethasone–trimethoprim liquid formulations.

further studies are time to time made refining of the optimization with steroids and other compounds as the models.178 prediction of the RP-HPLC retention of steroids using solvatochromic parameters. Chromatographic data–topological index dependence of steroids in RP-HPLC and RP-TLC are also worth mentioning.g. was successfully coupled on-line with electrospray MS.170–172 the method was successfully used for the determination of spironolactone.160 This technique. among others steroids. great many papers have been published on this topic. e. the simultaneous determination of hydrocortisone 21hemisuccinate. high pH buffers for weakly acidic estrogens.177 the use of experimental design for spherical coordinate representations of solvent composition in RP-HPLC.2trifluoroethanol as a mobile phase component in ternary systems together with perfluorinated RP-HPLC stationary phases unique selectivity was attained explicable by the strong adsorption of the co-solvent on the surface of the stationary phase.181 the effect of the pH of the mobile phase for the separation of ionisable steroids.176 methyltestosterone.195 5 Electrophoretic and Related Methods 5·1 Capillary electrophoresis (CE) Being neutral compounds the overwhelming majority of steroid hormone drugs cannot be analyzed by direct CE.162 Using 2.199–201 Since then. Based on these. e.173.776 HPLC stationary phases were prepared among others for the Porous screening of corticosteroids158 and estrogens.164 Cyclodextrins are known to be excellent chiral selectors in enantiomeric analysis (see section “Enantiomeric analysis”). Evaporative light scattering detector can be of importance in the case of UV-inactive steroids such as pancuronium bromide and The native fluorescence of related compounds. ethoxynonafluorobutane was introduced as an environmentally friendly and highly selective solvent in the NP-HPLC analysis of several compounds.196 derivatization with electrically charged reagents.198 5·2 Micellar electrokinetic chromatography (MEKC) MEKC is an ideal tool for the separation and quantification of lipophilic steroid drugs which cannot be directly analyzed by CE: these (mainly corticosteroids) were among the first models investigated after the invention of this new technique at the mid 1980s. The selectivity of the separation in RP-HPLC was improved by using ternary systems containing methyl or ethyl acetate as demonstrated by the separation of 9αfluoroprednisolone acetate from its impurities. too.2. VOL. oxytetracycline and nystatin in pharmaceutical Covalent preparations was carried out at pH 11. e. the identification and quantitation of as low as 0.197 Capillary electrophoresis can be a useful tool for the preconcentration of different classes of steroids. HPLC is a useful tool in other fields of steroid drug research.168 corticosteroids. termed “electrochemically modulated liquid chromatography”.and 17oxosteroids can easily be separated and quantificated.1 M sodium dodecyl sulfate (SDS) as the micelle forming agent was applied to steroid analysis by Spanish groups. In addition to retentionstructure relationship studies169 and screening of steroids.205 prednisolone.202 Some of the several new applications of MEKC to pharmaceutical steroid analysis are listed here: the determination of ethinylestradiol and levonorgestrel203 or gestodene204 in oral contraceptives.159 graphitic stationary phases were also used for the separation of corticosteroids especially if a voltage is applied to the column. This technique is restricted to ionizable derivatives.g. These include the description of a general quantitative relationship for column selectivity in RP-HPLC and the study of the effect of the change in conditions. borate for the complexation of steroids with proximal hydroxyls.161 Several papers have been published dealing with the mobile phase selection and optimation in the HPLC analysis of steroids hormone drugs.185 derivatization followed by electrochemical detection for the determination of spironolactone–hydrochlorothiazide tablets186 ANALYTICAL SCIENCES MAY 2004. ketosteroids with Girard P or T reagents is a solution for this problem: the Girard hydrazones of 4-ene-3-oxo.183 The first example for the improvements in the selectivity of the UV detection in the HPLC analysis of steroids is the detection of co-eluting species in e.01% prednisolone On-line post-column photochemical in hydrocortisone.182 the effect of column overloading on the separation of mometasone furoate and clotrimazole at widely different concentrations. Zn- ..g.. a special branch of RP-HPLC where the eluent contains very low concentration of organic modifiers (mainly 1-pentanol and acetonitrile) and about 0.193 Calculations based on linear solvation energy relationship were found to be useful to find “open windows” for internal standards in RP-HPLC chromatograms of various compounds.189 lipophilicity profiling.. danazol by subtracting the up-slope and down-slope diode-array spectra from the apex spectrum.184 HPLC-MS is an excellent tool for estimating minor components under overlapping peaks. For example HPLC was used for the determination of apparent association constants of steroid-cyclodextrin inclusion complexes. For example. 20 or diode-array UV detection for various drugs including corticosteroids in creams187 can be considered to be curiosities. clotrimazole and their related substances as the model compounds.170 In addition to its analytical application. among others steroids194 and these calculations were used to find suitable internal standard for the determination of estradiol and levonorgestrel in transdermal drug delivery formulations.167 The use of micellar liquid chromatography.180 triangle optimization for the separation of finasteride and related compounds.g.163 Another fluorinated solvent. while making use of its electric conductivity. Although the principles and practice of mobile phase optimization in HPLC were already set up in the early period of the history of HPLC. micelle forming agents for hydrophobic steroids. making use of electroosmotic flow and capillaries partially filled with various reagents.192 micellar chromatography with polyoxyethylene (23) lauryl ether (Brij35) as the micelle forming agent was used for the prediction of human drug absorption. hydrocortisone and its acetate together with various antibiotics. low costs and high speed MEKC can be a real alternative to HPLC in steroid analysis as demonstrated in a comparative study using betamethasone dipropionate. it is predictable that due to the minimum requirements of sample pretreatment. The effect of β-cyclodextrin on the retention of steroids and their separation in general165 and the effect of temperature on these characteristics166 were thoroughly studied.188 ethinylestradiol (ex: 285 nm/em: 310 nm) is used in the fluorimetric detection of the USP dissolution test of levonorgestrel–ethinylestradiol tablets7 while terbium(III)sensitized fluorescence (ex: 245 nm/em: 545 nm) detection was described for various steroids in the course of their SDS micellar HPLC determination.168–176 This technique enables the relatively rapid analysis of derivatives with wide range of polarity with minimum need for sample preparation.174 anabolic steroids176 and danazol175 in various formulations.190 for studying drug-membrane interactions191 and Biopartitioning high-throughput log P measurements.179 the use of 23 factorial design and computer simulation.

Of the innumerable studies. In the majority of cases differential pulse polarography.209 A comparison of the two systems using corticosteroids as the model compounds is also described. based on the reduction of the 4ene-3-oxo group is used. identification/structure elucidation and quantitative determination of impurities and degradants usually require the complex application of one or two separation method and at least 2 – 3 spectroscopic techniques (usually UV.243 (mainly products of intramolecular oxido-reduction reactions were identified in the course of the stress stability test of mazipredone (21-deoxy-21-N-methylpiperazinyl derivative of prednisolone).199. termed hydrophobic interaction electrokinetic chromatography seven active ingredients of an ointment (among them hydrocortisone) were separated and quantitated.249 oxidative degradation products of these250 and of nestorone239 are described.000 plates per meter was achieved. testosterone.220 With an inhouse packing method of capillaries up to 190. 20 bacitracin and phenylephrine in ointments and ocular drops. The performance of HPLC. MS. among them pancuronium bromide. this method does not seem to have been used as extensively in the practice as MEKC.214 In some further studies aiming at comparing the selectivities of the two techniques among many other compounds some steroids were also included. Z and E isomers of the 17-(3′acetoxy-2′-butenoate)251.241 and degradants242.238 Impurity and degradation profiling i.245. sulfacetamide and phenylephrine in local pharmaceutical preparations. 5·3 Microemulsion electrokinetic chromatography (MEEKC) Although in an early study better separation characteristics were attained for steroids with MEEKC than with the related technique MEKC. In the course of the impurity profiling of estradiol HPLC-UV played predominant .218 MEEKC is a good tool for the rapid estimation of log Pow values of a wide range of compounds.221 High-temperature CEC and temperature programming resulted in a considerable reduction of the separation run time of steroids.206 prednisolone acetate.252 and 17-(3-oxo-butenoate)1 identified in ethynodiol diacetate are by-products of the acetylation of the 17-OH group. In this study SDS or glycodeoxycholate were used as the micelle forming agent in MEKC and various higher alcohols.208 betamethasone in dissolution tests. for e. MEKC and CEC was compared using six steroids (androst-4ene-3. progesterone and the 17-OH and 20-OH derivative of the latter). VOL.17-dione. Impurities found in allylestrenol originate from its total synthesis.224 tipredane and related impurities225 and fluticasone propionate and related impurities226 is mentioned. respectively. Among minor impurities the 9α-bromo analogue of the parent drug was found as the main impurity in triamcinolone acetonide.215–217 A good correlation was found between the retention characteristics of betamethasone and its esters and the respective log Pow values. the 17β-carboxy-17α-formyloxy derivative forming under storage conditions via a mixed anhydride is especially interesting. unpredictable structures.235 A direct potentiometric method was developed for muscle relaxant quaternary ammonium compounds.g. NMR) either off-line but preferably on-line.246 Of these.246 The structure elucidation of impurities in fluticasone propionate (among others an interesting –S–S– bridged dimer) is a good example for the application of new techniques such as CEC-MS228 and on-line The light-induced degradation of HPLC-NMR. n-hexane.210 High concentrations of organic solvents (up to 50% acetonitrile or methanol) in the background electrolyte containing SDS enables ionic (benzalkonium chloride) and nonionic (beclomethasone dipropionate. In a few cases.211 Using a related method. 2-phenylethanol) compounds to be determined within one electrokinetic run. and the latter may be a good alternative to solvent programming. among them steroid hormone drugs.236 7 Selected Analytical Tasks 7·1 Impurity and degradation profiling of steroid drugs237.209 In the above-listed studies (and the majority of those not mentioned here) sodium dodecyl sulfate (SDS) was used as micelle forming agent in the background electrolyte. Six synthesis-related impurities were identified in prednisolone.222 Usually ODS phases are used for steroid separations but the successful use of macroporous. norgestrel) by-products of their ethinylation step. based on ion-selective membrane electrodes prepared from ion pairs with tetraphenylborate or dipicrylaminate as the counter ions in a PVC matrix. bile salts were also used.248 Of the (contraceptive) gestogens (norethisterone. cyclohexane.e. has been successfully applied to separation and quantitation of steroid drugs.232 dexamethasone sodium phosphate233 and betamethasone valerate.ANALYTICAL SCIENCES MAY 2004.229 6 Electroanalytical Methods A review of electroanalytical methods in steroid analysis was published in 1989.231 beclomethasone dipropionate. An example for chiral CEC is presented in the section “Enantiomeric analysis”. spherical polystyrene-divinylbenzene stationary phase was also described.227 propionate228 and the combination of CEC with nanospray MS with detection limits of 50 fg for corticosteroids merit special attention.213 Chiral aspects of MEKC are shown in the section “Enantiomeric analysis”. The migration behavior of the hydrophobic and ionic constituents was influenced by the concentration of tetradecyl ammonium bromide and ammonium chloride. which can be considered to be the combination of RPHPLC and CE.239 Several synthesis-related impurities240.240 Two estrogens are also worth mentioning. trimethoprim and polymyxin B in various formulations.234 Gestodene was determined in oral contraceptives by square-wave voltammetry and adsorptive stripping techniques. etc.247 methylprednisolone suleptamate lead to interesting. spironolactone. a few resulting in interesting structures are listed here beginning with corticosteroids. The very promising possibilities of the new technique CEC- 777 MS have been reviewed with several examples from steroid The identification of impurities in fluticasone drugs. detection.212 On-line sample preconcentration (370-fold improvement in detector response) was achieved for steroids by sweeping with anionic-zwitterionic micelles.207 dexamethasone.230 These methods have never played very important role in this field but sufficiently sensitive and selective methods are often published up to the present time mainly for the assay of formulations. Higher peak efficiencies were obtainable by MEKC and CEC. norethisterone.240.244 Of the other corticosteroids several synthesis-related impurities and degradants were identified in dexamethasone. with SDS as the emulsion-forming solvents.223 Of the practical applications the rapid analysis of norgestimate and its degradants.219 5·4 Capillary electrochromatography (CEC) and CEC-MS CEC.

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United States Pharmacopoeia 26. which is mainly administered as enantiomerically pure levonorgestrel.C. N. Microchim. Chichester. 1.. 30.253 while HPLC-NMR and HPLC-MS were necessary to determine the structures of five most interesting structures (a hydroperoxide and four different dimers) in the course of oxidative and light-stress studies of ethinylestradiol. Castañeda. 1999. C. J. R. Guzmán Bernardo. Blackie. Castañeda. M. Council of Europe. Biomed.778 role. 1054. 2.5-dimethylphenylcarbamate) were successfully used for CEC separation of the enantiomers. etc. Anal. Acta. Makin . J. J. D. 32. “Analysis of Steroid Hormone Drugs”. 257. 30. Anal. estrone. Korkmaz. 20. 39. C. C. several impurities originating from the Birch reduction step were identified in norethisterone. and A . “Ultraviolet-Visible Spectrophotometry in Pharmaceutical Analysis”. J. Castañeda. Andrisano. N. 1976. VOL. Anal. J. Rockville. The Japanese Pharmacopoeia 14th Edition. 7. G. J. 82. 2002. Görög and Gy. 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For this reason many papers contain data for norgestrel. Pérez Arroyo. “Colorimetric and Fluorimetric Analysis of Steroids”. Pharm. Elsevier. and G. Görög. Chim.250 an isomeric impurity in danazol255 and several oxidative degradation products in tipredane. Chim. M. 437. 41. E. usually among many other model compounds. Anal. 2001.264 and covalently bonded β-cyclodextrin. J. and J. 19. European Pharmacopoeia 4. J. Rodríguez Flores. Lett. Pharm. Pharm. Görög. Lett. Lett. E. 30. 32. Pharmeuropa. Arias. S. 6. S. 2002. Anal. S. For this reason enantiomeric analysis is not necessary in the overwhelming majority of cases. NMR spectroscopy using γ-cyclodextrin273 or the lanthanide shift reagent praseodymium tris[3-heptafluoropropylhydroxymethylene)-(+)-camphorate]260 was found to be suitable for estimating the enantiomeric purity of levonorgestrel without separation. A. J. The limit of detection is somewhat higher than that achievable by HPLC. 1997. J. 31. L. Berzas. 1999. London. Shively and A. 29. 2002. 10. Elsevier. J. El-Saharty. Anal. .. Nepote. Biomed. J.1% dextronorgestrel was achieved. Lett.. Y. M. Pesez. and also (at a decreasing extent) as the racemate. I.271 A comparison of the latter in the HPLC and CEC modes revealed significant advantages of CEC. Murat Palabıyık. 141. 1995. 122. 11. 55. USP Convention Inc. 16. Strasbourg. Atmaca. Analyst. J. Castañeda. 340. 2001. E. The only important exception is the total synthetically prepared norgestrel.. Luis. L. E. and F.. 13. Jimenez. I. Pharm. and F.. Tatar and S. 15. M.5-dichlorophenylcarbamate) and amylose tris(3. Amsterdam. ANALYTICAL SCIENCES MAY 2004. G. 883. P. Üstündag˘. Hassan.267 SFC using immobilized polysiloxane-anchored permethyl-β-cyclodextrin (Chirasil-Dex)268 and teicoplanin or its aglycone (Chirobiotic T or TAG)269 was also used to separate the enantiomers of norgestrel. Several papers have been published on the separation of the enantiomers of norgestrel. ethinylestradiol.260.265–267 even bonded to a monolithic silica column. Satuf.225. Rodríguez. Anal. R. 1989.. Toral. Garcia.258 (With β-cyclodextrin fairly good separation is achievable at 0˚C only. J. 1983. Anal. Richter. 374. 2000. 8.. Chim. D. their configuration is fixed in natural steroids and the semi-synthetic drugs prepared from them. 1029. 20 8 References 1. 22. Szász. 2001. Lemus Gallego and J. F.. and M. 34. 29. 2003. Society of Japanese Pharmacopoeia. Lemus Gallego and J.). J. and V. Santoro. J. 85. 2003. 1995. 32. An oxidative degradation product243. J. T. H. Acta. U. 2002. and A. 2003. M. and J. 1997. Biomed. Olivieri. 12. 247. G. Rodríguez. F.251 and synthesis-related impurities were identified and quantificated by NP-HPLC. J.132 NP-ion-pairing HPLC257 and the oxidative degradant also by quantitative NMR spectroscopic measurement. Erk. and D. Collado. M. A. Gatti.. Chem.272 In addition to this. Bonazzi. S. JAOAC Internat. 50. Tapia. Gianottto. In the first validated method RP-HPLC with γ-cyclodextrin in the mobile phase was used and a limit of quantitation of 0. 621. Glasgow. Í. M. Gazdag. B. S. 1997. Arias. and S. 85. Berzas. J.260 Norgestrel is an excellent model compound for testing the separation efficiency of various enentioselective chromatographic and related techniques. 1996. Anal. thus creating the possibility to find a correlation between the NMR and RP-HPLC data. Acta. 18. 460. 460. Lett. 31. 133. “Quantitative Analysis of Steroids”. Babják.263. 2002.5-dimethylphenylcarbamate). P. J. 4.

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