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tion enzymes (delta 4-hydrogenase, steroid dehydro- genase, hydroxylase, etc.), hence conjugated steroids are targets of direct analysis by high-performance liquid chromatography (HPLC)–MS which is de- scribed in more detail later. The most commonly used type of MS is the negative chemical ionization type.
3.2.1.2. Free form
Free forms of steroids in urine are often at too low
a level (e.g., cortisol about 100 nM/day) to assay, but represent the active form of steroids in serum. Therefore, enrichment of free urinary steroids are carried out by extraction of urine specimens with octadecyl silica (ODS, C18) or BSA-ODS (bovine serum albumin bound ODS) gel packed minicolumns prior to the assay of free steroids [9,10].
3.2.2. Serum
Steroids in serum specimens exist in three forms,
(i) free form (about 4%), (ii) albumin bound form (about 5%) and (iii) globulin bound form (about 91%). Only free forms of steroids are active bio- logically, hence the albumin and globulin bound forms are inactive.
3.2.2.1. Free form
Steroids in free forms are under negative feedback
regulation by pituitary (adrenocorticotropic hormone; ACTH, follicle stimulating hormone; FSH, lutenizing hormone; LH) and hypothalamic hormones (cortico- trophin-releasing hormone; CRH, LH releasing hor- mone; LH-RH, FSH-releasing hormone; FSH-RH), and show circadian rhythms of excretion. To obtain information of serum-free steroids, serum specimens are ultra filtrated by dialysis [11] or membrane filtration.
3.2.2.2. Globulin bound form
A large part of unconjugated serum steroids are
bound to serum globulin. Sex steroid hormones (androgens, estrogens and progestins) bind to globulin (sex hormone binding globulin, SBG) [12] and corticosteroid hormones also bind to globulin (corticosteroid binding globulin, CBG) [13]. These steroids bound to globulin have no biological activity as steroids. Binding force of steroids to SBG or CBG
is strong, but the steroids are released by extraction with organic solvent or a C 1 8 gel minicolumn [14].
3.2.2.3. Albumin bound form
Free steroids bind to albumin in serum [15], and
steroids bound to albumin have no biological activi- ty. The binding force of steroids to albumin is weak, and albumin bound steroids are easily released from albumin with organic solvents (e.g., dichlorome-
thane, ethyl acetate, and diethyl
ether) and
C 1 8
gels.
3.2.3. Saliva
Unconjugated steroids in saliva are present in the
free form, and reflect level of free steroid in serum with good correlation. Therefore, free salivary ster- oids assays are carried out for the purpose of obtaining information of free steroids in serum [16,17]. Free salivary steroid assay is easier than free serum steroid assay because of non-invasive and convenient sampling and less interfering substances.
3.2.4. Hair
Steroid assay in hair has not been popular in the
clinical laboratory until now. However, determina- tion of steroids in hair has advantages of easy conservation of hair specimens, no circadian rhythm of steroid excretion, and retrospective measure of steroid excretion. There are some reports on anabolic steroid [18,19], dehydroepiandrosterone [18], es- trogen [20,21], progesterone [20] and corticosteroid [22] assay in hair. The steroids in hair specimens were extracted after alkaline digestion, then deter- mined by GC or HPLC coupled to MS [18,22].
4. Recent progresses in separation analysis of steroids
Steroid analysis has been carried out for medical diagnosis of stress, hypertension, Cushing syndrome, adrenogenital syndrome, amenorrhea, and infertility, etc. For assaying steroid in human body fluids, there are additional requirements in comparison with assays in other fields [23,24]. Practical considera- tions are listed as follows.

4.1. Rapid assay
Separation assay by GC, HPLC or capillary elec- trophoresis (CE) takes a long time (e.g., 40–60 min) per specimen, and preparation procedures for speci- mens such as extraction or derivatization add more time to the assay. For clinical assay of steroids, a few minutes of assay time per specimen are required.
4.1.1. Fast HPLC with a microbore column
Sizes of column and gel used influence analysis time of steroids. Recent developments of column and gel size tend to down sizing, e.g., a microbore and short column (3–5 cm long, 1–2 mm I.D.), gel size (1–2 mm) [25]. Smaller and shorter columns packed with smaller sized gels brought more rapid analysis times within 5 min per specimen with no loss of resolution capacity [26]. Reversed-phase HPLC
using a C 1 8 column is popular for steroid analysis. 4.2. Highly sensitive assay
As steroids in human fluids are sometimes at too low levels to detect, highly sensitive determination methods are required. Fluorescence and chemilumin- escence detection methods for steroids were de- veloped for highly sensitive assays.
4.2.1. Fluorescence
Fluorescence detection methods for steroid assay
were employed to assay of steroids at low levels. As steroids have no native fluorescence except for estrogens, derivatization of steroids was required. Although the derivatization and its clean up pro- cedure have problems of time consumption for clinical assay, some inventions to solve the problem were developed (e.g., on-line photochemical deri- vatization of estrogens, simple sulfuric – ethanol de- rivatization of corticosteroids). The fluorescent meth- ods for steroids analysis reported were native fluores- cence, photochemical derivatization, sulfuric acid – ethanol fluorescence, and fluorescent derivatization with labeling reagents.
4.2.1.1. Native fluorescence of estrogens
Only estrogens among steroids emit native fluores- cence (emission 310 nm), and this allows rapid and relatively specific analysis of estrogens [8]. How-
ever, the native fluorescent intensity of estrogens is not strong because of its short emission wavelength of 310 nm.
4.2.1.2. Photochemical derivatization
Estrogens were converted to fluorophores by on-
line UV radiation post-column, and assayed by fluorescence detection (excitation 280 nm, emission 312 or 410 nm) [27]. This photochemical derivatiza- tion of estrogens had no need of clean up procedures after derivatization, and enabled rapid and sensitive determination of estrogens.
4.2.1.3. Sulfuric acid–ethanol fluorescence derivati- zation
Cortisol, corticosterone and testosterone, which are non-fluorescent, were converted to fluorophores (excitation 365 nm, emission 520 nm) by incubation with a mixture of concentrated sulfuric acid and ethanol for a few minutes without heating. The intensity of fluorescence is strong, and detection limit of cortisol was 0.26 mg/dl (S/N53). This sulfuric acid – ethanol derivatization method was relatively specific to cortisol, corticosterone, cor- tisone, dexamethasone and testosterone, and used originally for determination of 11-hydroxycorticoste- roids (11-OHCS) in serum or urine [28,29] (Table 1). Then the sulfuric acid–ethanol derivatization method was applied to the assay of cortisol and corticosterone by HPLC with fluorescence detection [30–32] (Fig. 1). This enabled highly sensitive and simple detection of cortisol, corticosterone and testo- sterone, but needed careful dealing with concentrated sulfuric acid (Table 1).
4.2.1.4. 9 - Anthroylnitrile derivatization of cortico- steroids
Corticosteroids are generally difficult compounds for derivatization because of its no active functional groups. However, 21-hydroxyl group of corticoste- roids was targeted for fluorescent derivatization with 9-anthroylnitrile (9-AN) [33,34]. The pre-column derivatization of corticosteroids with 9-AN needed clean up procedures prior to HPLC separation.
4.2.1.5. Dansyl derivatization of estrogens
The phenolic group of estrogens was derivatized with dansyl chloride to fluorophores off-line at
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