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The Vitamin D External Quality Assessment Scheme (DEQAS) was launched in 1989 and monitors the performance of 25-hydroxyvitamin D (25-OHD) and 1,25- dihydroxyvitamin D (1,25(OH) 2 D) assays. In April 2015 a pilot scheme for 24,25-dihydroxyvitamin D (24,25(OH) 2 D) was introduced. The 25-OHD scheme is accuracy − based with target values assigned by the NIST Reference Measurement Procedure (RMP) for 25-OHD 2 and 25-OHD 3 . A similar method is used to assign values for 3-epi-25-OHD. Five samples of human serum are distributed quarterly to over 1000 participants in 58 countries (April 2016) and clinical laboratories are expected to submit results within approximately 5 weeks. Research laboratories with assays run less frequently are not given a deadline. Archived samples with NIST- assigned values are also available. Performance is assessed on the first four samples with the fifth reserved for investigations e.g. recovery experiments or to assess the influence of other serum constituents such as lipids. DEQAS provides rapid feedback, with an on-line preliminary report available immediately after a participant submits results and a comprehensive report soon after the results deadline. In 2015, DEQAS investigations revealed that several 25-OHD immunoassays under-recovered 25-OHD 2 and 25-OHD results were falsely low on a sample with a modestly raised triglyceride concentration. An RMP for 1,25 (OH) 2 D is not yet available and results are judged against the Method Mean. Free advice is available from the DEQAS Advisory Panel which includes experts on methodology and biostatistics. DEQAS collaborates closely with the Vitamin D Standardization Program (VDSP) and both organizations have successfully worked with participants and manufacturers to improve the accuracy of vitamin D assays.

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes.

The Vitamin D External Quality Assessment Scheme (DEQAS) has been monitoring 25-OHD assay performance since 1989. The scheme has expanded rapidly in recent years and has 670 participants in 35 countries (July 2009). Five samples of human serum are distributed quarterly and the results analyzed to give an All-Laboratory Trimmed Mean (ALTM) and SD. Each participant has internet access to a preliminary report after submission of results and, following the results deadline, a final report is e-mailed to designated staff in each laboratory. The last 15 years has seen an improvement in mean inter-laboratory imprecision (CV), from 32.0% (1994) to 15.3% (2009) and most major methods are now giving results within plus or minus 7.4% of the ALTM (2009). DEQAS has regularly conducted and reported on a number of investigations into the performance of 25-OHD methods. A gas chromatography-mass spectrometry (GC-MS) reference method for 25-OHD is under development and will be used to assess whether the ALTM remains the most appropriate target for DEQAS samples.

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH)(2)D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within +/-30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D(3) or Vitamin D(2) was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH)(2)D were done 'on demand' and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.

Homogenates of histologically normal human testis from three men were incubated separately with pregnenolone, 16-dehydropregnenolone, 5alpha-pregnane-3,20-dione, 3beta-hydroxy-5alpha-pregnan-20-one and androsta-5,16-dien-3beta-ol (androstadienol) in the presence of NADPH in a study of androst-16-ene and androgen biosynthesis. After the addition of internal standards and initial extraction and purification, metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and monitoring selectively for three principal ions in each case at the appropriate GC retention time. Quantification was achieved by comparison with calibration lines for authentic steroids, together with the appropriate internal standards, prepared by monitoring three ion fragments for each analyte. In all experiments, androstadienol was found to be the major androst-16-ene metabolite of pregnenolone (seven times the control, i.e. endogenous, quantity; 19.8 +/- 3 ng/100 mg homogenate protein, mean +/- SEM, n = 9). Pregnenolone was also converted to androsta-4,16-dien-3-one (androstadienone) with three times the endogenous quantity (44 +/- 10 ng/100 mg homogenate protein, mean +/- SEM, n = 9) being formed. The formation of testosterone occurred only in trace amounts in the incubations of testis taken from one man (a 69-yr-old) but appreciable yields (six times endogenous levels 90 +/- 7 ng/100 mg homogenate protein, mean +/- SEM, n = 9) were found with testes from two younger men. Only traces of 5alpha-dihydrotestosterone were detected. Using androstadienol as the substrate, androstadienone was shown to be the major metabolite (approximately 10 times greater than control incubations) together with 5alpha-androst-16-en-3alpha- and 3beta-ols at approximately twice the endogenous quantities (5 ng/100 mg homogenate protein). In some incubations with androstadienol, 5alpha-androst-16-en-3-one (5alpha-androstenone) was formed (32 +/- 1 ng/100 mg homogenate protein/h; mean +/- SEM, n = 3); surprisingly, no endogenous 5alpha-androstenone could be detected. No evidence was obtained for the production of testosterone or 5alpha-DHT from androstadienol. Using cytosolic fractions of human testis, specific (displaceable) binding of 5alpha-androstenone was determined, with binding sites of approximately 200 fmol/mg tissue and a Ka of approximately 8 nmol/l.

A case is described of a 37-year-old man who was breath tested by the U.K. police following a traffic accident and was found to have a breath-alcohol concentration of 70 micrograms/100 mL--twice the U.K. legal limit. The defendant protested vigorously that he had not consumed sufficient alcohol to account for this excessive reading, and Widmark calculations, assuming his account of the alcohol consumed was accurate, suggested his contention was possibly correct. No evidence was obtained suggesting that the Lion Intoximeter used for the breath analysis was anything other than accurate. Alcohol loading tests were therefore carried out in the laboratory on two separate occasions, showing that breath-alcohol concentrations grossly in excess of the legal limit (140 micrograms/100 mL) were obtained when the amount of alcohol administered would have been expected to give a theoretical maximum of 35 micrograms/100 mL. A blood sample taken at the point when the breath-alcohol concentration was 70 micrograms/100 mL was shown to contain 54 mg of alcohol/100 mL of blood. Dental examination of the defendant showed that he had had extensive work carried out, including three bridges. A possible explanation, therefore, for these anomalous results is that the excessive breath-alcohol concentrations might be due to mouth alcohol retained in the bridges or periodontal spaces, although a careful fraud by the subject of this report cannot be ruled out.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of indole-3-carbinol (IC), an anticarcinogen present in cruciferous vegetables, to alter the metabolism of 4-androstenedione (AD) by female rat liver microsomes was investigated and compared to that of its main gastric conversion product, diindolylmethane (DIM) as well as other specific cytochrome P450 inducers. DIM was a more potent inducer of the hydroxylase which converts androsterone to its 6 beta-hydroxylated derivative 3 alpha, 6 beta-dihydroxy-5 alpha-androstan-17-one (A) than IC after either oral or intraperitoneal administration and was also a better in vitro inhibitor. Isosafrole (ISF), which like IC and DIM, induces CYP1A2 as well as gestodene, were powerful inhibitors of the in vitro reaction. Naringenin produced only a weak inhibitory effect while 3-methylcholanthrene was inactive. SKF-525A, a prototypic hydroxylase inhibitor, or 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androst-1-ene-3-one which inhibits steroid 5 alpha-reductase, also decreased the formation of A from AD by liver microsomes. The infusion of human growth hormone by osmotic minipump, which feminizes hepatic steroid metabolism, increased the ability of male rat liver microsomes to convert AD to A and to respond to induction by IC. The identity of A, the main polar derivative of AD, induced by IC, DIM and ISF, was tentatively assigned by a combination of GC-MS and results from metabolic studies with intermediates in the pathway leading to its formation. It is proposed that the protective role of indole carbinols against mammary carcinoma due to decreased formation of 16 alpha-hydroxyestrone from estrone may be further enhanced by the diminished availability of AD for aromatization to estrone.

Steroids consist of an essentially lipophilic (or hydrophobic, non-polar) cyclopentanoperhydrophenanthrene nucleus modified on the periphery of the nucleus or on the side chain by the addition of hydrophilic (or lipophobic, polar) groups. Although steroids are widely distributed in nature and many thousands have been synthesised in the laboratories of pharmaceutical and chemical organisations, this chapter concentrates primarily on the methodology for the analysis of steroids of biological importance to human subjects and in particular on the methods for the analysis of the very low concentrations of steroids found in human biological tissues or formed during in vitro or in vivo studies. This does not, however, imply that the techniques discussed here may not find applicability in other areas of steroid analysis. This chapter neither discusses specifically the saturation analysis techniques including immunoassay-radioimmunoassay (RIA), enzymeimmunoassay (EIA), which are explained in Chapter 4, nor the analysis of cardenolides, sapogenins, alkaloids, brassinosteroids or ecdysteroids, which present their own analytical challenges but are of less interest in a clinical context. Further details on basic principles of mass spectrometry (MS) are discussed in Chapter 2.

The analysis of vitamins D and their metabolites and analogues has been reviewed by us on two occasions (Makin et al., 1995; Jones and Makin, 2000) over the last 10-15 years. In this chapter, we have drawn heavily on the 2000 review, up-dating it to take account of the developments in methodology that have occurred in the intervening years, but including elements of our 1995 review so that the reader can get a picture of the historical context as well as the modern developments.

Biologists use the word ‘estrogen’ when referring to molecules which have the ability to induce uterine growth or vaginal cornification in the immature or ovariectomized rodent. The word estrogen was derived from two Greek words – oistros meaning frenzy and gennein – to beget. Chemists and biochemists, however, often restrict their use of this term to molecules that contain a characteristic 18-carbon steroid nucleus with an aromatic (phenolic) A-ring, both those that are biologically active estrogens and those without biologic activity but which are of intrinsic interest, such as the estrogen conjugates. This chapter is concerned only with these steroid compounds. The structure and inter-relationship of some common estrogens are given in Fig. 8.1. In addition to the biological estrogens, there are a wide variety of both natural and synthetic compounds which have estrogenic activity when measured by one or another parameter. While many of the assay procedures described in this review are applicable to these compounds, their application to non C18-steroids will not be discussed here. Methodology for these non-steroidal compounds can be found in reviews by Wang et al. (2002), Wu et al. (2004), Muir (2006), and Delmonte and Rader (2006). While not wishing to downgrade the importance of previous work in the estrogen field, the authors have taken a deliberate decision to exclude most publications prior to 1975, not because these do not have value but simply because space is not unlimited and readers of the present chapter might be expected to be seeking information about methodology which is less than 30 years old. Readers seeking pre-1975 information in this area can find it in Oakey and Holder (1995).

The adrenal cortex is the source of a number of steroid hormones that play a fundamental role in regulating body metabolism. The elucidation of the complicated biosynthetic pathways and the metabolic fate of these hormones proceeded relatively slowly until it became possible to make reliable measurements of the concentrations of these important steroids in body tissues. Because of the complex structure of the steroids, their close chemical similarity to each other and their occurrence in low concentrations in body tissues, the development of suitable analytical techniques proved a major challenge to chemists and biochemists working in this field. Although many of these problems still exist, there has been significant progress in the clinical field, particularly because of the availability of radiolabelled and stable isotope-labelled steroids and the introduction of immunoassay (IA) methods for routine clinical biochemical analysis. The advent of highly selective separatory chromatographic methods (LC and GC) increasingly linked to mass spectrometers means that analytical methods are now available for all of the important adrenal cortical hormones, their major precursors and metabolites, and synthetic glucocorticoids. These methods have found wide application, especially in clinical research and investigation, where they have been used to elucidate the nature of many endocrine disease processes, and for diagnosis and treatment. These facts in themselves highlight the importance to the analyst of ensuring that the techniques used are as reliable as possible.

The development of novel gene expression systems for cytochrome P450s (CYPs) together with a revolution in analytical mass spectrometry with the emergence of liquid chromatography/mass spectrometry (LC/MS) has opened the door to answering some long-standing questions in Vitamin D metabolism. Our studies focused on: (1) elucidating the role of CYP24 in 25-OH-D 3 and 1α,25-(OH) 2 D 3 metabolism; (2) exploring how DBP influences this process; (3) measuring 25-OH-D 3 metabolism in CYP24–knockout (CYP24–XO) cells and; (4) comparing 1α-OH-D 2 metabolism in the CYP24-XO mouse in vivo and in vitro. Methodology employed CYP24 over-expression and knockout systems in conjunction with state-of-the-art analytical LC/MS, diode array, and radioisotopic detection methods. We found that CYP24 metabolizes 25-OH-D 3 and 1α,25-(OH) 2 D 3 at similar rates in vitro, but that for 25-OH-D 3 but not 1α,25-(OH) 2 D 3 , this rate is strongly influenced by the concentration of DBP. Unlike their wild type littermates, the administration of 25-OH-D 3 to CYP24-XO mice results in no measurable 24,25-(OH) 2 D 3 production. When neonatal murine keratinocytes are prepared from wild type and CYP24-XO mice there was no measurable production of 24,25-(OH) 2 D 3 or 1α,24,25-(OH) 2 D 3 in CYP24-XO mice. Similar experiments using the same wild type and CYP24-XO animals and cells and [ 3 H ] 1α-OH-D 2 resulted in the apparent paradox that the Vitamin D prodrug was 25-hydroxylated in vivo but 24-hydroxylated in vitro.

Vitamin D*, a secosteroid derived from a sterol precursor by opening of the steroid B ring between carbons 9 and 10, exists in a number of different chemical forms. In the clinical environment, only vitamins D2 and D3 are important. Vitamin D3 (cholecalciferol) arises by the action of UV light on the sterol precursor, 7-dehydrocholesterol, which is found in the upper layers of the skin. The chemical reactions involved are shown in Figure 10.1. It can be seen that apart from the production of the provitamin and subsequent conversion into vitamin D3, a number of other isomers are formed. The proportion of these different products from 7-dehydrocholesterol varies depending upon the conditions under which they are formed. Vitamin D2 (ergocalciferol) arises in a similar fashion in that it derives from the UV irradiation of the 5,7-diene sterol precursor, ergosterol. Ergosterol is widely distributed in plants, yeasts and a number of animals other than humans. Vitamin D2 has an extra methyl (C-28) attached to carbon 24 on the vitamin D side-chain together with an extra double bond at C-22(23), and its structure is given in Figure 10.1. Because the further metabolism of vitamin D involves hydroxylations on the side chain, it is inevitable that the physiological effects of vitamin D2 and vitamin D3 may well be slightly different. For historical reasons, vitamin D2 is more readily available commercially and is used in a number of countries throughout the world to supplement milk and other foodstuffs, and calciferol BP is in fact vitamin D2.

Human semen was examined for the presence of 16-androstenols, 16-androstenones and androgens. Extracts were analysed by gas chromatography-mass spectrometry after derivatization of steroids under study. In a qualitative study, 5 alpha-androst-16-en-3 alpha- and 3 beta-ols, 5,16-androstadien-3 beta-ol and 5 alpha-androstan-3 beta-ol were detected in a semen pool A. Hydroxyl groups were converted to tert-butyldimethylsilyl ethers, the ions selected for monitoring being [M-57]+, consistent with loss of the tert-butyl group. For a more detailed quantitative study, a second semen pool B was used. In this case, all hydroxyl groups were converted to trimethylsilyl ethers, while oxo groups were not derivatized. As with semen pool A, separation of steroids was achieved using capillary gas chromatography with appropriate temperature programming. Quantification was carried out by mass spectrometry using selected ion monitoring of two significant ions and appropriate internal standards. The following steroids were identified at the concentrations indicated: 5 alpha-androst-16-en-3 alpha- and 3 beta-ols and 5,16-androstadien-3 beta-ol (concentration range, 0.5-0.7 ng/ml). 5 alpha-Androst-16-en-3-one and 4,16-androstadien-3-one were also present at levels of 0.7-0.9 ng/ml. Two androgens, testosterone and 5 alpha-dihydrotestosterone were found at concentrations of 0.5 and 0.3 ng/ml, respectively. These data, showing the presence of 16-androstenes and androgens in human semen, appear to be consistent with testicular formation of these steroids. The possible significance of the odorous 16-androstenes is discussed.

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT 2 and DHT 3 . Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT 2 ) was demonstrated in human plasma after administration of oral DHT 2 . Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT 3 in vitro showed that 25-hydroxy-DHT 3 was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT 3 -23,26-lactone and 23,25-dihydroxy-24-oxo-DHT 3 . The osteosarcoma cell line metabolized 25-OH-DHT 3 in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1α,25-dihydroxy-DHT 3 . The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1α,25-dihydroxy-DHT whereas compound Hb is possibly 1β,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1α,25-dihydroxy-DHT is very effective in displacing radiolabelled 1α,25-dihydroxyvitamin-D 3 and is thus most likely to be the calcaemic metabolite of DHT.

The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC-further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered-methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.

A patient with an apparent abnormality of cortisol binding globulin (CBG) is reported. Investigations showed that while the plasma CBG binding capacity (equivalent to the plasma CBG concentration) was normal, the binding affinity was reduced by a factor of four. The significance of this observation is discussed with regard to its possible role in the development of the adrenocortical hyperfunction also seen in this patient.

A high-performance liquid chromatographic (HPLC) method for estimating plasma 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3) is described. The method involves plasma extraction, Lipidex 5000 chromatography and HPLC on straight-phase Zorbax-SIL, collecting the 25-OHD2 + 25-OHD3 fractions. These secosteroids are isomerised to their isotachysterol derivatives and re-run in the same HPLC system, monitoring at 290 nm. 3H-Labelled 25-OHD3 is used as an internal standard. The method was evaluated in terms of reproducibility, and recovery of added secosteroids was quantitative. Values obtained using this method were in close agreement with those values obtained on the same plasma sample using gas chromatography—mass spectrometry.

Saliva collected from volunteers of both sexes at various times throughout the day was examined for the presence of 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 ,. The method used was a specific high-performance liquid Chromatographic procedure with a minimum detectable limit for 25-hydroxyvitamin D of 0.05 μg/1, (130 pmol/1 for 25-hydroxyvitamin D 3 ) using 20 ml of saliva. No 25-hydroxyvitamin D could be detected in any of the samples. Examination of larger quantities of saliva (100 ml) by a combination of high-performance liquid chromatography and gas chromatography-mass spectrometry showed the presence of a peak possibly derived from 25-hydroxyvitamin D 3 , at a concentration not in excess of 65 pmol/1. Using known binding characteristics of 25-hydroxyvitamin D and plasma vitamin D binding protein, and comparing them with similar characteristics for cortisol and plasma cortisol binding globulin and the known salivary concentration of cortisol, simple calculations indicated that a likely concentration of 25-hydroxyvitamin D in saliva would be around 8 pmol/1. Previously reported values ranging from 130–4160 pmol/1 (50–1600 μg/l) using a simple competitive protein binding assay could not be confirmed.

A new method for the extraction of bile acids from human plasma using acetonitrile precipitation of plasma protein and subsequent use of Bond-Elut C18 cartridges is described. After extraction the bile acids can be separated into three fractions: unconjugated, glycine-, and taurine-conjugated, using Sep-Pak SIL cartridges at 4 degrees C, eluting with ethanol--chloroform--water--glacial acetic acid mixtures. These extraction and fractionation procedures were evaluated in terms of recovery, reproducibility and resolution between the fractions. All these parameters were found to be satisfactory. Although the reproducibility of fractionation on Sep-Pak SIL cartridges was found to vary between batches, this did not give rise to significant difficulties. Plasmas from normals and patients with hepatobiliary disease were analysed by capillary gas-liquid chromatography after extraction and fractionation using the procedure described.

A simple polarographic oxygen electrode is described which has been adapted to provide a portable apparatus for the specific estimation of blood glucose using the enzyme glucose oxidase. The method is capable of giving accurate, single estimations of the glucose concentration in 0.05 ml of venous or capillary blood or plasma within 2 min. The apparatus is designed for use by non-technical staff in hospital wards or outpatient departments where rapid information about blood glucose levels may be of value.

A rapid gas-chromatographic method for the fractionation of the major 17-oxosteroids and 17-oxogenic steroids in urine is presented. The method utilises borohydride reduction, periodate oxidation and gas-liquid chromatographic (GLC) separation of steroid formate esters on SE 30. A single estimation may take less than 4 h, providing estimates of the urinary excretion of pregnanetriol and THS (together); allopregnanetriol and allo-THS (together); aetiocholanolone; androsterone; THE, THF, cortols and cortolones (as a group); allo-THE, allo-THF, allo-cortols and -cortolones (as agroup); pregnanediol; 11-oxygenated aetiocholanolone; 11-oxygenated androsterone. The method has been evaluated by comparing 17-oxosteroid (17-OS) and 17-oxogenic steroid (17-OGS) values calculated from the individual GLC values with those obtained by group estimations on the same urine. The correlation coefficients between the GLC values and the group values did not differ significantly from 1.000 (17-OS, 0.979; 17-OGS, 0.976). Recovery experiments using reference steroids and available steroid glucuronides indicated quantitative recoveries. Steroid sulphates were not measured. Reproducibility was of the order of 10% (coefficient of variation). The specificity of the method was examined by trapping GLC peaks which were. subjected to mass spectroscopy and chromatographed in other GLC systems. In only one case so far examined has a GLC peak proved to be other than the expected steroid formate.

Publisher Summary This chapter discusses the use of hydroxyapatite in radioimmunoassay. Any radioimmunoassay analysis relies upon the efficiency of the separation of the unbound (free) antigen or hapten from that bound to antibody. When using immunoassay to estimate low molecular weight compounds, such as steroids, the problem is somewhat simpler than in protein immunoassay. Because of the large difference in molecular weight between steroid haptens and their appropriate antibodies, relatively simple methods of separation, such as dialysis, can be used. This chapter describes the experiences in the radioimmunoassay of steroids, using hydroxyapatite as an adsorbent, for the steroid-antibody complex. Hydroxapatite has proved to be very useful for the separation of free and antibody-bound steroid in the immunoassay of various steroids. Successful immunoassays have been set up, using hydroxyapatite, for the estimation of plasma testosterone, estradiol-17β, 17α-hydroxyprogesterone, and progesterone. No steroid, so far examined, has been found to be bound to hydroxyapatite. Some other low molecular weight compounds, for which immunoassays are available, have also been found not to be bound to hydroxyapatite, and it may, therefore, be possible to use hydroxyapatite in immunoassays for compounds other than steroids.

A simple procedure for the esterification of steroid carboxylic acid groups using an acetyl chloride/alcohol reagent has been carefully evaluated. The ease with which these esters can be formed using this reagent depends on the acid and the ester required. C24 bile acids form methyl esters using acetyl chloride/methanol (1:20, v/v) almost instantaneously at 50 degrees C, whereas the C20 aetienic acid studied requires 2 h at 50 degrees C. The formation of isopropyl esters of 5 beta-cholanic acid takes longer (4 h at 50 degrees C) than the formation of n-propyl esters (1 h at 50 degrees C). The identity of various esters fo cholanic acids formed using this reagent have been checked by gas chromatography-mass spectrometry and various other procedures used for the methylation of fatty acids have been examined to establish whether they can be used for the quantitative esterification of bile acids.

A simple one step high-performance liquid Chromatographie (HPLC) procedure for the analysis of plasma concentrations of 25-hydroxyvitamin D 3 (25-OHD 3 ) and 25-hydroxyvitamin D 2 (25-OHD 2 ) is described. Plasma (2–4 ml) was extracted with methyl cyanide which was passed through a Sep-Pak C 18 cartridge, washed with methanol: water (70:30, v/v) and the 25-OHD fraction eluted with methyl cyanide. After isomerisation to their isotachysterol derivatives, the secosteroids were estimated using a straight-phase HPLC system, monitoring the eluent at 301 nm. Radioactive 25-OHD 3 , added to plasma at the start of the procedure, was used to correct for losses. Recovery of added 25-OHD 3 was quantitative and values obtained using this method were similar to those obtained on the same plasma samples using a mass fragmentographic technique. Normal ranges were similar to those described by other workers and within- (5.8% for 25-OHD 3 ) and between- (8.0% for 25-OHD 3 ) batch reproducibilities were satisfactory.

A mass fragmentographic assay for vitamin D 3 , 25-hydroxyvitamin D 3 , 25-hydroxyvitamin D 2 and 24,25-dihydroxyvitamin D 3 is described. After extraction of plasma with methanol and dichloromethane, Lipidex 5000 chromatography was used to separate the plasma extract into three fractions (a vitamin D fraction, a 25-hydroxyvitamin D fraction and a 24,25-dihydroxyvitamin D fraction). Cholesterol was removed from the vitamin D fraction by thin-layer chromatography. After addition of vitamin D 2 to each fraction, isotachysterol trimethylsilyl ethers were formed. Recoveries prior to gas chromatography-mass spectrometry were assessed by the use of tritiated standards added to the plasma before extraction. Using this procedure, normal ranges were established (vitamin D 3 : 3–17.7, 25-hydroxyvitaminD 3 : 5.9–35.2, 25-hydroxyvitamin D 2 : 0.6–1.0 and 24,25-dihydroxyvitamin D 3 : 0.6–2.9 μg/l).

The results of a forensic toxicological investigation on a young man with an unknown cause of death are reported here. Analysis revealed the presence of a possibly fatal level of terodiline in blood and urine. No other drugs were detected. Terodiline was detected by thin-layer and gas-liquid chromatography and identified by gas chromatography/mass spectrometry. Quantification was carried out by a mass fragmentographic procedure using the m/z 100 from terodiline for selective ion monitoring (SIM). The blood and urine concentrations were found to be greater than 10 mg/L, whereas therapeutic concentrations in serum are usually not more than 1 mg/L. Support and confirmation of the laboratory results was provided at the subsequent inquest. It was revealed that the deceased had died from the inhalation of vomit due to an oral overdose of terodiline. To the best of our knowledge this is the first reported death due to fatal poisoning with terodiline in the United Kingdom.

Over the last two decades, interest in the metabolism and physiological action of the group of secosteroids collectively known as “vitamin D” has increased exponentially [l-4]. In 1968 Blunt, DeLuca and Schnoes[5] isolated and characterized a new metabolite of D3t, 250HD3, which was more effective than the parent vitamin in the cure of rickets, stimulation of intestinal calcium transport and in the mobilization of bone calcium. Further studies by separate research groups led to the discovery of a number of dihydroxylated vitamin D metabolites, 24,25(OH)2DJ [6], 25,26(OH)2D3 [7] and 1,25(OH),D, [8-l 11. Of these, 1,25(OH),D, is the most potent form of vitamin D known, being many times more active than vitamin D in the stimulation of intestinal calcium transport, the mobilisation of calcium from the bone and in the prevention of rickets [8,9, 12,131. One of the hurdles to a greater understanding of the importance-of vitamin D in physiological systems has been the difficulty in measuring levels of vitamin D and its metabolites in normal and abnormal states. There are many reasons for this; the low level at which the metabolites circulate in plasma, the number of different metabolites and their chemical similarity to one another and their similarity to other neutral lipids such as cholesterol and finally the instability of these secosteroids to ultraviolet light, and heat [see 41.

The first description of successful capillary gas chromatography of intact glycine-conjugated bile acid derivatives used an automatic solid injection system and required very high carrier gas flow-rates (approximately 20 ml/min) to obtain satisfactory peak shape. Peak heights of unconjugated bile acid derivatives using this injection system and the low flow-rates (1-2 ml/min) usually used for such gas chromatographic analyses were very susceptible to small changes in flow-rate. This system has been re-examined in an attempt to explain this anomalous behaviour. An alternative injection system, the all-glass dropping needle, was also investigated. No explanation for the very high carrier gas flow-rates required when using the automatic solid injection system was found. The dropping needle injection system, however, produced excellent separation of bile acids and their glycine conjugates as dimethylethylsilyl ethers-methyl esters on non-polar wall-coated capillary columns using normal carrier gas flow-rates of 1-2 ml/min. It is clear that the automatic solid injection system originally used has some problem associated with it which can only be overcome, in the case of bile acids and their glycine conjugates, by increasing the carrier gas flow-rate to very high levels.

This review attempts to provide a concise and critical summary of modern methods for the analysis of bile acids and their conjugates in human biological fluids. Most emphasis is given to more up-to-date procedures that have been applied to the study of human disease and attention is drawn to previous reviews in areas that have not been covered here. An increasing awareness of the possibility that bile acids may be involved in the etiology of a number of disorders, or that such disorders may give rise to changes in bile acid concentration, has stimulated the study of bile acid methodology. Although many procedures have been described using, for example, high-pressure liquid chromatography (HPLC), gas-liquid chromatography (GLC), gas-liquid chromatography--mass spectrometry (GLC-MS), and radioimmunoassay (RIA), no simple but comprehensive procedure for the estimation of bile acids and their conjugates has yet been published. Further study in this area is still required in order to establish the role of bile acid estimations in the routine diagnosis and treatment of disease.