ANALYTICAL SCIENCES MAY 2004, VOL. 20 767 2004 © The Japan Society for Analytical Chemistry Reviews 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 767 4·3 Supercritical fluid chromatography (SFC) 2 Steroid Hormone Drugs in Pharmacopoeias 768 4·4 High-performance liquid chromatography 2·1 Assay of bulk drug materials (HPLC) and HPLC-MS 2·2 Related impurities test of bulk drug materials 5 Electrophoretic and Related Methods 776 2·3 Assay of steroid hormone formulations 5·1 Capillary electrophoresis (CE) 3 Spectroscopic and Spectrophotometric Methods 772 5·2 Micellar electrokinetic chromatography (MEKC) 3·1 Ultraviolet-visible spectrophotometry 5·3 Microemulsion electrokinetic chromatography 3·2 Fluorimetry (MEEKC) 3·3 Infrared and Raman spectroscopy 5·4 Capillary electrochromatography (CEC) and 3·4 Mass spectrometry CEC-MS 3·5 NMR spectroscopy 6 Electroanalytical Methods 777 3·6 Chemiluminometry 7 Selected Analytical Tasks 777 3·7 Circular dichroism (CD) spectropolarimetry 7·1 Impurity and degradation profiling of steroid 4 Chromatographic and Hyphenated Methods 774 drugs 4·1 Planar chromatography 7·2 Enantiomeric analysis 4·2 Gas chromatography (GC) and GC-MS 8 References 778 development in steroid hormone drug analysis during the last 15 1 Introduction years can be characterized by the facts that HPLC undoubtedly became the most important routinely used method and that the The aim of this paper is to give an overview of the advances importance of hyphenated chromatographic–spectroscopic during the last 15 years and of the state-of-the-art in the analysis techniques is increasing. New techniques, mainly capillary of steroid hormone drugs and related materials. Why just 15 electrophoresis (CE) and related techniques, such as micellar (or years? My first book on steroid hormone drug analysis was microemulsion) electrokinetic chromatography (MEKC, published in 19782 when this area was in transition state due to MEEKC), capillary electrochromatography (CEC), also attract the introduction and rapid spreading of new techniques, mainly wide interest in steroid analysis. Summarization of the results high-performance liquid chromatography (HPLC) and achieved with these methods will be the subject of this review. immunoassay methods. This was followed by another book in Only hormone drugs, their semi-synthetic analogues and 19833 dealing with wider areas (in addition to the analysis of hormone-like structures are considered. Other groups of hormone drugs other types of steroids and biological-clinical steroids (sterols, bile acids, cardiac glycosides, vitamins D, etc.) aspects). The third book, published in 19894 (just 15 years ago), are not within the scope of this review. Pharmaceutical- dealt mainly with pharmaceutical and industrial aspects. The industrial aspects are discussed, such as assay and impurity profiling of bulk drugs and drug formulations, estimating This paper is Part 56 in a series on “Analysis of Steroids”; for degradation profiles. Biological-clinical aspects, Part 55 see Ref. 1. pharmacokinetic and metabolic studies are also not within the E-mail: [email protected] scope of this review. These aspects are dealt with in a book of 768 ANALYTICAL SCIENCES MAY 2004, VOL. 20 Makin et al.5 Spectroscopic studies are not discussed either, pancuronium bromide6,8 and stanozolol6,7 with acetous unless these are part of impurity profiling and degradation perchloric acid. The lactone group in oxandrolone,7 after profiling studies. opening it with sodium hydroxide and the –O–SO2–ONa group in sodium prasterone sulfate8 after Na+ → H+ ion exchange, are also suitable for titrimetric determination. 2 Steroid Hormone Drugs in Pharmacopoeias Of the chromatographic methods other than HPLC, gas chromatography is only very seldom used for the assay of bulk Pharmacopoeias are naturally rather conservative: new steroid hormone drugs. The outdated and labor-consuming techniques appear in their monographs only if proved by many method of TLC separation and UV spectrophotometry after spot years of practice that the performance of these methods is elution is prescribed for the assay of meprednisone, nandrolone superior to that of the currently used methods. In spite of this phenylpropionate, prednisolone hemisuccinate and limitation, the state-of-the-art of methodology and requirements testosterone.7 in official drug analysis is well reflected by monographs concerning the latest revisions of the principal pharmacopoeias. 2·2 Related impurities test of bulk drug materials The test for “Related substances”,6 also named 2·1 Assay of bulk drug materials “Chromatographic purity”,7 “Ordinary impurities”,7 “Related Table 1 shows a brief summarization of the assay and related steroids”7 or “Other steroids”,8 is the most important test to impurities tests in the monographs of bulk steroid hormones and characterize the quality of a bulk drug material, much more hormone-like structures in European Pharmacopoeia 4th ed. important than the assay, especially if the latter is carried out by (Ph. Eur. IV),6 United States Pharmacopoeia 26th ed. (USP non-specific methods. XXVI)7 and the 14th edition of the Japanese Pharmacopoeia As can be seen in Table 1, with two exceptions (conjugated (Ph. Jp. XIV).8 and esterified estrogens, where gas chromatography is used), As can be seen in Table 1, the most frequently used assay this test is carried out in all cases by semi-quantitative TLC or method in Ph. Eur. IV. is UV spectrophotometry at about 240 quantitative (mainly but not exclusively reversed phase) HPLC nm for steroids with strongly absorbing 4-ene-3-oxo-or 1,4- methods. The proportion of HPLC tests for related impurities in diene-3-oxo group and at about 280 for estrogens with a phenol- bulk steroids in various pharmacopoeias is quite different: about type ring A with its weak, but characteristic, spectra. This 75% in Ph. Eur. IV, 50% in USP XXVI and 10% in Ph. Jp. method is naturally non-specific: the overwhelming majority of XIV. It is amazing that in many cases the USP XXVI and Ph. the impurities are measured together with the main component. Jp. XIV do not contain tests for related impurities (27 and 10%, The predominant method in the United States Pharmacopoeia is respectively). highly specific reversed phase (in some cases normal phase) In the case of HPLC tests, a UV detector is exclusively used at HPLC. In the overwhelming majority of cases, porous the wavelength of the maximum of the main component or at octadecylsilica support (3 – 10 µm) is used for the RP and 254 nm. For the thin-layer chromatographic purity test in the porous silica support (5 – 10 µm) for the NP separations. The majority of cases, sorbent layers are used that are impregnated wavelength of the UV detector is set most often at 254 nm, with dyes strongly fluorescing when irradiated at 254 nm by a which is not the maximum of the spectrum of 4-ene-3-oxo- or mercury lamp. Since most of the steroid hormone drugs 1,4-diene-3-oxosteroids. This is attributable to the early period strongly absorb UV light at this wavelength, the drugs and their in the history of HPLC when the majority of instruments were impurities appear as dark spots in the chromatogram suitable for equipped with only a mercury lamp. In the Japanese their semi-quantitative estimation. These appear as “TLC 254 Pharmacopoeia the HPLC and UV spectrophotometric assay nm” in Table 1. In some cases the use of various visualizing methods are almost equally represented. It is to be noted that reagents is necessary. Reagents containing sulfuric acid are UV spectrophotometry around 240 nm with limits for the most frequently used. “TLC–H2SO4” in Table 1 means spraying specific absorbance is often part of the monographs as an with this reagent, heating and visual inspection of the plate and identification test, even in those cases when the assay is carried the use of long-wavelength UV light (366 nm). Other out by HPLC methods. visualizing reagents are phosphomolybdic acid It is interesting to note, and difficult to explain that in some (TLC–phosph.mol.), p-toluenesulfonic acid, vanillin/sulfuric instances visible spectrophotometric methods, such as a acid (TLC–vanillin), potassium dichromate/sulfuric acid measurement around 380 nm after condensation of the (TLC–acid dichromate), iodine vapor (TLC–I2) and alkaline unsaturated 3-oxo group with isoniazide, or an indirect Blue Tetrazolium (TLC–tetrazolium). With a few exceptions, measurement at 525 nm after a reaction with Tetrazolium Blue both the TLC and HPLC tests express the impurities as the main of corticosteroids with reducing side chain, is prescribed by the component, which can be source of serious errors (under- or European and United States Pharmacopoeias; moreover, in one overestimation) if the chromophoric system or the color and/or instance (mestranol)7 the assay is based on a non-stoichiometric the intensity of the TLC spot of the main component and the color reaction with a methanol–sulfuric acid reagent (545 nm).