Steroid profile

Que AH, Palm A, Baker AG, Novotny MV. 2000. Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray-mass spectrometry. J Chromatogr A 887(1-2) 379-391. 

Kushnir et al. [44, 45] also measured 17-hydroxyprogesterone within a profile of four adrenal steroids (detailed in Hormonal Steroid Profiles below). They utilized an oxime derivative to improve ESI sensitivity and carried out analyses on an ABI 4000 instrument. Rauh and co-workers [70] published a method for 17-hydroxyprogesterone with ancillary measurement of androstenedione and testosterone. The mass spectrometer was an ABI 4000 instrument with an APCI source, operated in positive-ion mode and with on-line cartridge sample extraction and column switching. The MRM transitions were as used by other workers (Table 5.3.1)... 

Corticosterone is a component of the steroid profile (see Hormonal Steroid Profiles , below) developed by Guo et al. [25]. The analysis uses positive APPI and a d8 corticosterone internal standard. MRM was conducted with the m/z 347—>121, and 355—>125 transitions, respectively. The lower detection limit (LOD) was 2 pg/ml. Quest Diagnostics offer MS/MS analysis of this steroid commercially and have published normative values, as listed in Table 5.3.1. Marwah et al. [56] have published a method for the quantitation of corticosterone in rat plasma using electrospray MS. MS/MS methods for deoxycorticosterone (DOC) have not been published. 

CAH caused by 11/Thydroxylase deficiency is diagnosed by finding elevated 11-de-oxycortisol. This steroid is included in the profiles of Guo et al. [24,25] and Kushnir et al. [44] described below in Hormonal Steroid Profiles . Guo et al. monitored the transitions m/z 347—>97 for the analyte and 349—>97 for the dideutero internal standard. Kushnir et al., utilizing the oxime derivative monitored transitions 371—>24 and 112 for the analyte and 379—>124 and 112 for the dideutero standard. Quest diagnostics offer this MS/MS method and provide most of the quantitative values listed in Table 5.3.1. 

These steroids are components of the profile of Kushnir et al. [44, 45] and are discussed below in Hormonal Steroid Profiles . They were analyzed as the oxime derivatives by ESI on an API 4000 instrument. The MRM transitions for pregnenolone oxime were 332—>86 and 300, and for the d4 internal standard 336—>90 and 334. Transitions for 17HP were 348—>330 and 312, and for the d3 internal standard 351—>333 and 315. The publication gives quantitative data for Tanner stage 1-5 male and female children and for several age groups up to 52 years. 

Measured as part of a CAH panel by Minutti et al. [58], using the transition 287—>97, see above ("17-Hydroxyprogesterone ). They stress the added specificity of CAH detection in second-tier analysis for CAH when androstenedione is measured as well as 17-hydroxyprogesterone. This is also measured commercially by Quest Diagnostics and included in the Guo et al. hormonal profile [24, 25] (see Hormonal Steroid Profiles below). 

I consider hormonal steroid profiles to be those analyses that encompass a panel of steroids that can diagnose different endocrine abnormalities. A panel of multiple steroids used to diagnose a single condition, for example 21-hydroxylase deficiency, would not be a steroid profile under this definition. 

Guo and co-workers [24,25] have spearheaded the development of MS/MS serum steroid profiles. Their most recent report describes profiling in 11 min of 12 steroids in 200 pi serum with minimal work-up, comprising acetonitrile protein precipitation. The steroids analyzed were as follows DHEA sulfate, DHEA, aldosterone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, estradiol, testosterone, 17-hy-droxyprogesterone, progesterone, and 25-hydroxyvitamin D3. Stable-isotope-labeled internal standards were incorporated for each steroid. An API-5000 instrument was used with the APPI source in positive-ion mode, with the exception of aldosterone, which had greater sensitivity in negative-ion mode. Separation was carried out on a C8 column, which allowed more rapid separation than the more commonly utilized C18. The MRM transitions utilized are shown in Table 5.3.1. The lower level of sensitivity was between 1.5 and 10 pg/ml, dependent on the steroid. The authors were exhaustive in addressing issues of accuracy, recovery (90-110%) and reproducibility (< 12.2% for same-day and between-day). 

The synthesis of adrenal steroids and major excreted metabolites is illustrated in Fig. 5.3.1. Little secreted steroid product is excreted unchanged and most of the catabolism takes place in the liver, although cortisol metabolism by the kidney is clinically important and microbial metabolism in the gut can be quantitatively significant. The major metabolic transformations of hormonal steroids and precursors are detailed by Makin [54] and summarized in Fig. 5.3.2. GC-MS steroid profiling is the technique of choice for measurement of important urinary constituents. 

It is in the neonatal period that steroid profile analysis is most useful for CAH diagnosis because serum 17-OHP assays can give elevated results, even for nonaffected babies, due to lack of specificity of the RIAs used [105]. This is becoming less of a problem with the increasing use of HPLC-MS/MS for 17-OHP measurement as... 

The THEiTHF + 5aTHF ratio is commonly greater than 15 in these disorders, the opposite to AME syndrome. While the ratio of saturated steroids demonstrates a high 11-oxo l 1/1-hydroxy ratio, this is not the case for steroids retaining a - -4-ene structure. The F E ratio is normal or elevated (i.e., > 1 Table 5.3.9). Steroid profile analysis also typically reveals an elevated excretion of DHEA and other androgen metabolites. Clearly the excessive ACTH production resultant from an apparent cortisol deficiency is responsible for the elevated adrenal androgen production, which in turn is responsible for female virilization and other manifestations of polycystic ovary syndrome. 

Guo T, Chan M, Soldin SJ (2004) Steroid profiles using liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization source. Arch Path Lab Med 128 469-475... 

Holst JP, Soldin SJ, Tractenberg RE, Guo T, Kundra P, Verbalis JG, Jonklaas J (2007) Use of steroid profiles in determining the cause of adrenal insufficiency. Steroids 72 71-84... 

Lacey JM, Minutti CZ, Magera MJ, Tauscher AL, Casetta B, McCann M, Lymp J, Hahn SH, Rinaldo P, Matern D (2004) Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry. Clin Chem 50 621-625... 

Steroids hormones in clinical chemistry Immunoassay vs. GC-MS and LC-MS/MS derivatization vs. nonderivatization steroid profiles for newborns, adrenal insufficiency, prostatitis/pelvic pain syndrome, premature adrenarche, sera from smokers, metabolic diseases, diabetes, water contaminant, athletes donine. [3]... 

MO-TMS derivatives were also used for the quantitative analysis of steroid profiles in urine [371,372]. The procedure involves extraction of steroids and conjugates by means of XAD-2 resin, enzymatic hydrolysis, solvolysis with acidified ethyl acetate and washing with alkali. The derivatives are prepared as follows. The organic solvent is evaporated under a stream of nitrogen at 60°C and 100 jul of a solution of methoxylammonium chloride in dry pyridine (100 mg/ml) are added to the dry residue. The mixture is heated in a closed vial at 60° C for about 1 h, then the excess of pyridine is removed under... 

Detection and quantitation of steroids. The confirmation of the steroid content of a biological sample is a routine procedure in many GC-MS laboratories. The development of the human urinary steroid profiles ... 

In clinical studies, steroid profiling by GC-MS continues to play an important role [1]. However, LC-MS is started to be applied for various special applications. Some results in relation to breast and prostate cancer research, and neurosteroids are discussed below. 

Brady H A, Johnson N N, Whisnant C S et al 1997 Effects of oral altrenogest on testicular parameters, steroidal profiles and seminal characteristics in young stallions. 

Wudy SA, Homoki J, Wachter UA, Teller WM. Diagnosis of the adrenogenital syndrome caused by llp-hydroxylase deficiency using gas chromatographic-mass spectrometric analysis of the urinary steroid profile. Dtsch Med Wochenschr 1997 122 3-11. 

Lipson SF, Ellison PT. Comparison of salivary steroid profiles in naturally occurring conception and nonconception cycles. HumP, eprod 1996 11 2090-6. 

Feist, G., C.B. Schreck, M.S. Fitzpatrick and J.M. Redding. Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation. Gen. Comp. Endocrinol. 80 299-313, 1990. 

Buiarelli et al. (2004) extended the above analytical approach to many more related steroids when they published a method for the direct analysis of 15 urinary anabolic steroids in a single run, namely T, epitestosterone, dehydroepiandrosterone (DHEA), androsterone, etiocholanolone, their sulfates and their glucuronides (Figure 2,2), They extracted 2 mL of human urine by solid-phase extraction with methanol elution and reconstituted the residue in aqueous methanol in the presence of deuterated internal standards (da-epitestosterone glucuronide, [16,16,17-"H3 testosterone sulfate and [16,16,17-2H3]testosterone), then monitored, for example, mJz. 289-97 and 109 for T and epitestosterone, miz 367-97 for their sulfates, and m/z 463-113 and 287 for their glucuronides. The method does not achieve quantitation, but it allows the estimation of ratios, which makes it possible to monitor the urinary steroid profile, which is useful for monitoring the abuse of anabolic steroids. 

Similarly the steroid profile of umbilical cord blood is very unlike that in adult blood and is dominated by large quantities of steroids with the 3j8-hydroxy-A configuration and by estrogens and progesterone and their metabolites. Many of the compounds involved are unique to this period of life, and it has been necessary to develop special techniques for their measurement (C12, C13, E4, S9, SlO, Sll, S20, Z4). 

Shackleton, C. H. L., Charro-Salgado, A. L., and Mitchell, F. L., Urinary neutral steroid profile analysis in adults and infants. Clin. Chim. Acta 21, 105-118 (1968). 

Fig. 3.18. Representative urinary steroid profiles of a normal male versus a diabetic male. Reproduced from [268].

Although the present use of urinary steroid profiling techniques appears to be largely confined to biomedical research, their gradual acceptance in clinical diagnosis and preventive medicine (to establish the risk of biochemical endocrine disorders)... 

Meyer, H. H., Rohleder, M., Streich, W. J., Goltenboth, R., Ochs, A., 1997, Sex steroid profiles and ovarian activities of the female panda Yan Yan in the Berlin Zoo, Berl. Miinch. TierdrztL Wochenschr. 110 143-147. 

Disease Cause Change in urinary steroid profile... 

Steroids Biie acids Fatty acids Urinary acids Ketones Prostagiandins Oximes, TMS, t-BDMS ethers Methyl esters, TMS ethers Methyl esters, TMS, t-BDMS esters Methyl esters, TMS, t-BDMS esters Oximes Methyl esters, oximes, TMS, ethers Steroid profiles Liver disease, peroxisomal deficiencies Phospholipids, triglycerides Acidurias, diabetes Diabetes Healing processes, pain... 

Steroid profile analysis (simultaneous measurement of various types of steroids) plays an important role in the clinical evaluation of a number of common endocrine disorders in humans and animal models. LC-MS profiles of steroids in bovine adrenal cells, the rat brain, and human serum have been reported during the past five years (Table 7). 

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