Automated urinary steroid profiles by capillary column gas-liquid chromatography and a computing integrator

237

Clinica Chimica Acta, 79 (1977) 237-253 0 Elsevier/North-Holland Biomedical Press

CCA 8795

AUTOMATED URINARY STEROID GAS-LIQUID CHROMATOGRAPHY

PROFILES BY CAPILLARY COLUMN AND A COMPUTING INTEGRATOR

VERA FANTL * and C.H. GRAY ** Department of Chemical Pathology, King’s College Hospital Medical School, Denmark Hill, London, SE5 8RX (U.K.) (Received January 28th, 1977)

Summary 13 Urinary steroid metabolites have been determined by automated gasliquid chromatography with a 20-m glass capillary column and a computing integrator. Concentrations up to 2 mg/24 h computed by the integrator compare well with those obtained by peak height measurements. At higher concentrations discrepancies occurred, particularly for the Czl steroids where falsely low values were calculated using peak heights. Mean excretion by healthy males and females of seven steroid metabolites is presented.

Introduction Several methods [l-6] have been reported for the gas-liquid chromatog raphic separation of urinary androgens, progestogens and corticosteroids by glass capillary column. This paper describes the linkage of capillary column gasliquid chromatography, with automatic injection and temperature programming, to a computing integrator, permitting the separation and quantitation of urinary steroids. Materials and methods Steroid reference compounds were obtained from Steraloids, Croydon, England, and the Medical Research Council Reference Collection. &Glucuronidase (Pasteur) was obtained from Institut Pasteur Production, Paris; bis(trimethylsilyl)trifluoroadetamide (BSTFA) from Pierce Chemical Co., Rockford. Ill. Absolute ethanol was supplied by James Burrough Ltd., London; potassium * Present adress to where requests for reprints should be sent: Department of ClinicalEndocrinology. Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, WC2A 3PX U.K. ** Present address: Division of Clinical Chemistry, Clinical Research Centre. Harrow. HA1 3JU. U.K.

23%

acetate, chloroform and cyclohexane, all of laboratory reagent grate, by BDH, Poole, England and a hexane fraction low in aromatic hydrocarbons, general purpose reagent, by Hopkin and Williams, Chadwell Heath, Essex, England. The last three solvents were washed twice with concentrated sulphuric acid and then with water until the aqueous layer was free from sulphuric acid. After drying with anhydrous Na2S04 the solvents were distilled and the chloroform stabilised by the addition of 1% by volume of ethanol. The potassium acetate was stored over CaCl, in a desiccator. Standards An ethanolic solution was prepared containing (IS) cholesteryl butyrate tion.

of the ten steroids marked with an asterisk in Table I 20 pg/ml of each component. The internal standard was prepared in cyclohexane at the same concentra-

Hydrolysis and extraction of urine Urine collected over 24 h was diluted to 2 litres. Urine from children was measured and used undiluted. A sample of lo-20 ml was adjusted to pH 6-6.5 with 1 M NaOH. Two ml was pipetted into a Quickfit test tube (125 X 18 mm with a 14 mm socket), mixed with 30 ~1 P-glucuronidase Pasteur, an enzyme prep~tion from ~sc~eric~ia coli with high specific activity [7], and incubated for 1 h at 45°C. Anhydrous sodium sulphate (0.34 g) was then added through a funnel with shaking on a vortex mixer. When the solid was completely dissolved, 1 pg cholesterol butyrate was added as an internal standard and the urine extracted with 10 ml chloroform. After centrifuging, the aqueous phase and the precipitate at the interface of the two layers were removed by aspiration. The extract was washed with about 0.1 ml 1 M NaOH (3 drops) until the aqueous layer was colourless (usually two washes) and then with slightly more (4 drops) of distilled water until the wash was free of alkali. After each wash the extracts were centrifuged briefly so that the small quantity of aqueous phase could be more TABLE TRIVIAL

I NAMES

AND

ABBREVIATIONS

*Androsterone *Aetiochol~olone *Dehydroepiandrosterone *llfl-Hydroxyaetiocholanolone *Pregnanediol *Pregnanetriol Pregnenetriol Tetrahydro S Pregnanetriolone Tetrabydro DOC *Tetrahydro E *Tetrahydro F BOc%-Hexahydro S ItOP-Hexahydro S *Cortolone *corto1 Internal standard

A E DHA 1lOHE PD PT A5PT THS PTL THDOC THE THF HHS 2Ocu HHS 2Op

IS

3ol-Hydroxy-~!-androstan-17.one 3~-Hydroxy-SP-androstan-17-one 3&Hydroxyandrost-5-en-17-one 301.1lp-Dihydroxy-6P-androstan-17-one 5@-Pregnane-3% 2Owdiol 5@Pregnane_3ol, 1701,2Ocu-trio1 Pregn-&ene-3a. 17q 20wtriol 3cu,17cl, 21-Trihydroxy-5@-pregnan-20-one 3a, 17% 20or-Trihydroxy-5P_pregnan-1 l-one 3or,21-Dihydroxy-5@-pregnan-20-one 301,17cr,2l-T~ydroxy-5~-pregnan-l1,2(f_one 3a, ll& 17% 2l-Te~ahydroxy-6~-pregn~-2O-one 5@-Pregnane_3u, 17a. 2Oc~,2l+etrol &J-Pregnane-3ff,l7cu. 20&214etrol 3a, 17% 20~~.21-Tetrahydroxy-5fkregnan-11-one So-Pregnane-3% lip, 17a, 20a, 21.pent01 Cholesteryl butyrate

239

easily removed by aspiration. The chloroform extract was evaporated under a stream of nitrogen in a 3 ml vial at 40°C in a heating block. Traces of moisture were eliminated from the residues by storing them in a vacuum desiccator at least 1 h. Preparation of trimethylsilyl derivatives To each urine extract 5-10 mg potassium acetate and 100 ~.tlBSTFA were added. After heating at 60°C in an aluminium block for 1 h the BSTFA was evaporated at the same temperature under a stream of nitrogen. 0.1 ml of the standard steroid solution (2 ,ug of each component), 1 pg of internal standard and 2 pg each of other steroid standards such as tetrahydro S (THS) and pregnanetriolone (PTL) when necessary, were mixed and dried under nitrogen at 40°C and similarly silylated. The dried residues were dissolved in 50 ~1 hexane and samples (2 ~1) and standards (1~1) were injected into open-ended glass capillaries about 7 mm X 1 mm i.d. and allowed to dry. The automatic injection compartment could accomodate 24 capillaries but because of differences in injection conditions for the final samples (see below) not more than 12 samples of which the lst, 5th and 9th were standards, were analysed in one batch. Gas-liquid chromatography A Packard model 419 Becker gas chromatograph with a flame ionisation detector, automated solid sampling system, model 770, with electropneumatic signal converter, model 762, and a multilinear programmer model 758, was fitted with a 20 m X 0.3 mm i.d. glass capillary column coated with OV-101 (Jaeggi, Miihlau, Switzerland) and connected to a Spectra-Physics Autolab System 1, digital computing integrator (Spectra Physics, Autolab Division, Santa Clara, Calif.) and a 10 mV recorder (model TOBNl-L Toshin Electron Co. Ltd., Tokyo, Japan). Capillary column end fittings described below were supplied by Packard Instruments Ltd., Caversham, Reading, U.K. Helium carrier gas inlet pressure was 10 psi and hydrogen and air flows to the detector were 25 ml/min and 300 ml/min, respectively. The injection compartment consists of a stainless steel disc 47 mm diameter and 15 mm thickness, the circumference of which contains 24 holes diameter 3 mm to accommodate the open-ended glass capillaries. A stainless steel base plate with one hole 4 mm diameter, coincident with the injection inlet, supports the samples. The two are mounted on a spindle which rotates the disc, the base plate remaining stationary, allowing the samples to drop through the injection inlet, into a glass tube 140 mm long and 4 mm in diameter. The descent of the capillaries is stopped by a narrow constriction less than 1 mm in diameter 120 mm down the tube. The end of the capillary glass column is fitted to #the other side of the constriction as follows. A P.T.F.E. ferrule (i.d. 8 inch + & inch) through which the column end is passed, is contained in the hexagonal nut of a Swagelok male adaptor. Careful tightening of the nut compresses the ferrule which forms a gas-tight seal and holds the column end in the constriction. After the first two or three temperature programmed runs the hexagonal nut needs further tightening. Thereafter no detectable leakage of carrier gas may occur for several months. The outlet of the capillary column is similarly fitted into the bore of the quartz glass jet of the flame ionisation detector. This

240

system previously described by Shackleton and Honour [8] differs mainly in that these workers used P.T.F.E. sleeves to connect the ends of their capillary column to lengths of glass-lined steel capillaries at both injection and detector ends. With these sleeve connections Shackleton and Honour [S] observed carrier gas leakage within a single temperature-programmed analysis. The injection zone is kept at 210°C and the detector at 260°C. The upper injection compartment is kept at about 40°C with an aluminium, water-cooled block. The multilinear programmer (Fig. 1) consists of a two-dimensional circuit board with 30 columns each of 14 sockets. The period of each column is selected from a choice of 5 “time bases” and the maximum duration of a programme cycle is 90 min. Each of the 14 sockets in the vertical axis represents a programmable function. Seven of the column sockets represent rates of increase in temperature, i.e. 0.5, 1.0,2.5, 5.0, 7.5 and 30”C/min; a further four ‘B’, ‘C’, ‘D’, ‘E’ represent isothermal heating, cooling, reheating and resettings controls. Cooling is accomplished by automatic lifting of the oven roof to which the column is attached. The remaining three sockets are auxillary controls for starting equipment such as automatic samplers and integrators. Indicators lights along the board permit the stage of the programme cycle to be known. Pins were connected as shown in Fig. 1. A time base of 3 min was used. The sequence of timed events for an analysis are shown in Table II. System 1 is an electronic digital integrator capable of printing out stored retention times and areas of peaks as well as calculated data. It detects and inte-

Fig. 1. Multilinear

temperatureprogrammingunit.

241 TABLE

II

SEQUENCE TOGRAPHY Time

OF MULTILINEAR

Oven temperature

PROGRAMMER

(“C)

FUNCTIONS

Programmer

USED

FOR

GAS-LIQUID

CHROMA-

function

(mm)

0 3 6 48 65 66 69 12 75

145 160 175 217 260 260 Ambient 145 145

Oven temperature increased at 5’C/min Sample injected Oven temperature increased at l”C/min Oven temperature increased at 2.5’C/min Maximum temperature reached Oven door opened Oven door closed Programmer reset Sequence restarted

grates peaks using a change in level or slope of the signals from the chromatographic detector. The signals measured in mV are converted to a frequency which is counted continuously in 0.1 set time segments. Narrow peaks with large increases in slope require less sensitivity for detection than broad peaks and the slope sensitivity on system 1 is therefore adjustable. The data in the 0.1 set time periods is grouped into “data bunches” and a slope calculated by averaging the signals obtained within this group. This averaging process helps to filter out baseline noise. The number of 0.1~set samples in a data bunch can be varied. The system 1 manual states that 10 data bunches at the half-height of a peak provides the minimum noise with the minimum amount of peak filtering. The peak width at half-height for our work was obtained froma trace of the steroid standard derivatives using a fast chart speed. The width at half-height of the narrowest peak of interest was measured in seconds and found to be 7 set; this was entered through the keyboard. Thus each of the ten data bunches contained seven O.lset samples. During an analysis peaks due to solvent residues and other contaminants occurring before 10.0 min are integrated but not stored or printed out. This is accomplished by pressing an “integrate delay” control and entering 10.0 at the eliminates small keyboard. Use of a “minimum area” control automatically peaks. In the present work, those equivalent to less than about 2% full scale recorder deflection were not stored. Once analysis is complete and the internal standard cholesteryl butyrate is eluted reports are obtained automatically using the “pre-set” time control at 63.0 min, with the “reset time” control at 75.0 min the integrating and reporting cycle restarts as the next sample is injected. To commence automatic separation and quantitation of a series of samples the multilinear programmer is started and exactly 3 min later integration is commenced by depressing the “run” button. An internal standard method was used for quantitation. The retention times of internal standard and steroids to be quantitated were entered into the integrator memory, which stores this information as relative retention times, the IS having a value of 1.00. The expected variation of these retention times within a batch of analyses, which we found to be small (see results section) was

242

entered as 2%. Thus of a group of peaks appearing within 22% of a given relative retention time, that closest to the value calculated from the entered data would be recognised and quantitated by the integrator. Very large off-scale peaks, however, may have retention times up to 5% greater than expected and are therefore missed for quantitation. This is then carried out using the raw integrated data. From a c~ibration mixture consisting of compounds to be qu~titated and internal standard the integrator is able to calculate and store the areas of the peaks due to the internal standard and those of the components of the calibration mixture and to calculate their ratios. Since the standard mixture contained 2 pg of each steroid and 1 pg of internal standard the calibration factor for a given component is given by area of internal standard x --~~concentration of component area of component concentration of internal standard = area of internal standard x 2.00 area of component 1.00 A different calibration factor is calculated and stored for each component to be quantitated. The internal standard amount added to the sample was the same as that in the calibration mixture. Thus the amount of a component steroid S in an injected urine sample is given by

given in

a.wa by S sample X calibration factor x I area given by IS in sample The mtegratmg calculator by the manufacturer’s mode 2A provides the amount of the component as a percentage of the amount of sample analysed. The amount can then be corrected to absolute amounts by entering 100 into the “sample amount” key. If the 24-h urine has been diluted to 2000 ml, mg excreted per 24 h is automatically printed, but if there is no dilution, as with infant’s urine or if the 24-h volume is greater than 2 1, the calculation requires the entry of a scale factor: (total volume)/2000. When the manufacturer’s mode 2B was used with a scale factor, erroneous results were obtained. Results and discussion The initial temperature (145-160°C) and carrier gas flow (lo-13 psi) were such that androsterone (A), the first steroid of interest, was eluted at about 15 min. The sample was concentrated as a narrow band on the column using a fast initial temperature programming rate of S”C/min for 3 min. This improved the resolution of peaks with retention times less than about 20 min; later peaks were unaffected. Programming was then kept constant at l”C/min until the pregnanetriol (PT) peak appeared. At 48 min the rate of temperature increase was changed to 2.5”C/min. This ensured that the increase began before the elution of tetrahydro E (THE). The faster rate of temperature increase produced sharper corticosteroid peaks and permitted completion of the analysis within about 60 min. Fig. 2 shows the separation of the mixed steroid standard after derivative formation. Tetrahydro DOC and Tetrahydro S were also included in the mixture.

243

Fig. 2. Gas-chromatographfc separation of a standard steroid mixture after sWation. about 40 ng of each companent. Column: OV-101(20 m X 0.3 mm Ld.).

Peaks nWr%@nt

Table IIf shows the station times and eIution temperatures of 14 steroid standards. After 4 to 6 months use of the column retention times of steroid derivatives decreased gradually by about 4 min each as the stationary phase bled from the column. Resolution also decreased p~i~~~~y of A, E, DHA a_nd IlOHE where peaks began to tail. Before this stage was reached, the “wit&innbatch” coefficient of variation for the retention times of 10 steroid standards was 0,22-4X866% (n = 13). A comparison of precision using peak h&@&s and ~u~~~ areas was made, The precision of a new column was compared with that of an old column and the linearity of the detector response was investigated. A total of 13 injections of l-3 ~1 was made of a standard containing cholestql butyrate and the TMS derivatives of 10 different steroids in 50 ~1 cyclohexane. Steroidjcholesteryi butyrate ratios were oboe from peak heights and jn~~a~d areas (Table IV) on the new column and precision for A, E and DWA was better using integrated areas than peak heights, the reverse being found for IIOHE. On the old column where tailing peaks were formed with thest: four steroids, precision decreased ~onsjde~b~y using peak heighti but much less using integrated areas

244 TABLE III RETENTION

TIMES FOR DERIVED

Steroid A E DHA 1lOHE PD PT A5PT THS PTL THDOC THE THF Cortolone Cortol IS

STEROID

STANDARDS

OBTAINED

Elution temperature (“C)

Retention time bin)

215 219 222 225 225 235 237 245 250 257

17.8 18.9 20.9 27.3 34.3 42.5 45.6 47.3 48.6 48.1 52.4 53.2 56.4 58.2 60.8

USING SYSTEM

1

with the exception of 11OHE where no change was found. For the CzI steroids precision on the new column was improved using integrated areas compared with peak heights. However, precision on the old column for these steroids did not deteriorate using either integrated areas or peak heights in contrast to results observed for the Cl9 steroids. Successive injections onto the column were not identical since the used glass capillaries accumulated in the injection comp~ment. The first capillary injected gave noticeably greater peak heights for the first four components compared with subsequent injections, where the slightly longer time between the sample volatilising and reaching the column appeared to cause band broadening. Pregnanediol and steroids with a longer retention time were not susceptible to this effect. It would seem that compensation for less efficient sample injection occurs for components with a long residence time on the column. The broader peaks obtained for the first four steroids eluted, especially on the old TABLE IV STEROID/INTERNAL Steroid

STANDARD

RATIOS

New column

Old column

Peak heights

Integrated areas

Peak heights

Integrated areas

Mean

Mean

Mean

Mean

C.V. (%)

A E DHA 1lOHE PD PT THE THF c0rt010ne Cortol

1.55 1.60 1.55 1.15 1.42 1.13 1.16 1.39 1.33 0.97

10.7 9.4 6.3 7.0 6.4 6.8 3.5 3.2 6.8 2.8

C.V. (%)

1.92 1.92 2.06 1.83 2.33 1.97 1.95 2.17 1.88 1.13

4.0 2.7 4.3 11.5 2.6 2.4 1.9 3.5 2.4 2.6

C.V. (%)

1.02 1.10 1.07 0.67 0.86 0.86 1.06 1.07 1.25 0.82

23.2 20.1 22.4 23.6 5.2 6.6 2.6 1.9 6.7 2.4

C.V. (%b)

1.74 1.77 1.88 1.52 1.74 1.93 1.45 1.35 1.83 0.96

8.3 6.5 7.1 11.1 1.9 1.5 2.0 4.3 2.3 2.9

245

column where tailing was observed would account for the marked improvement in precision using integrated areas for these compounds. Standards containing 1,2,3 and 4 ,ug of each of 9 steroids with 1 E.cgof internal standard were used to investigate linearity of detector response by peak heights and integrated areas. Since a standard containing 2 pg of each steroid was used for quantitation of urine samples this was used as the reference value. For the three androgens linearity was good using both methods although a slight deviation at higher concentrations occurred using peak heights (Fig. 3). For the remaining steroids, using peak heights linearity tailed off considerably at the two higher concentrations. However, integrated areas still gave a good linear response for pregnanediol and pregnanetriol. Only for the corticosteroids was a significant but minor deviation from linearity observed at the higher amounts of steroid. However, it was notably less than that obtained using peak height measurements. Cholesteryl butyrate is most frequently used as internal standard for steroid profiles. It has the advantage of being eluted at the end of an analysis where minimum interference from urinary constituents is likely. Some published methods [2,5] include a second internal standard eluted at the beginning of an analysis. The mean peak height of the two internal standards is used to calculate peak height/internal standards ratios. However, the integrator can only quantitate data using a single reference standard. Moreover the selection of a suitable second internal standard which will not overlap with early eluting uri-

RELATIVE

Fig. 3. Calibration ratios (0 7). used.

CONCENTRATION

A) and peak height curves for 9 steroid standards using integrated area ratios (AStandards containing 1, 2. 3 and 4 pg of each steroid with 1 pg internal standard were

246

nary components is difficult. Thus 5a-androstane-3a ,170( diol, 3fl-hydroxy-5/Iandostan-17-one (epi-aetiocholanolone) and oestra-1,3,5(10)16-tetraen-3-01 have variously been used. Shackleton and Honour [8] adopted a different approach. They measured the ratio of peak heights to the height of the line joining two internal standards 5a-androstane-3a,l7adiol and cholesteryl butyrate. Table V shows a comparison of the coefficients of variation obtained for 13 injections of a derivatised standard mixture using this technique, with those obtained previously using the new column (Table IV). Androsterone was used as the early eluting internal standard. Apart from values for pregnanediol and pregnanetriol, using the method of Shackleton and Honour [8] a consistent improvement in precision was obtained for peak height calculations, particularly for aetiocholanolone. However, overall precistion was still not as good as that obtained using integrated areas. Seven 2-ml extracts of a random sample of urine were prepared and analysed as described in the method. A comparison of quantitated values for seven metabolites using peak heights and integrated areas was made (Table VI). As could be predicted from linearity studies with standards larger discrepancies occurred at the higher concentrations of steroids measured. In contrast to result obtained with the pure standards precision was slightly better using peak heights, rather than integrated areas. This could be due to the greater background noise and overlapping peaks present in chromatograms of urine extracts. Although there was usually little variation during a run of three standards and eight urine extracts, three standards were regularly included and peaks evaluated against the nearest, the later standards purging the column to avoid excessive contamination with less volatile urinary impurities. The improved linearity with integrated areas produced more accurate values at higher steroid concentrations, although some errors were introduced into the results for larger amounts of corticosteroids. No correction was applied since these errors were unlikely to exceed about 7%. TABLE

V

COEFFICIENTS BY

OF

VARIATION

FOR

STEROID/INTERNAL

STANDARD

RATIOS

CALCULATED

3 METHODS

Steroid

C.V.

m?)

Peak

heights

1 internal

Integrated

standard

2 internal Internal

standards

*

standard

4.0

A

10.1

E

9.4

3.1

DHA

6.3

5.3

4.3

1lOHE

7.0

6.5

11.5

PD

6.4

9.4

2.6

PT

6.8

7.3

2.4

THE

3.5

2.6

1.9

THF

3.2

2.6

3.5

Cortolone

6.8

4.6

2.4

Cortol

2.8

2.5

Cholesteryl

butyrate

* Calculated

according

Internal to the

standard method

2.1

2.5

Internal of Shackleton

areas

and

standard Honour

Internal (see

text).

standard

247 TABLE VI REPRODUCIBILITY OF 7 INDEPENDENT A RANDOM URINE SAMPLE Steroid

A E PD PT THE THF Cortolone

DETERMINATIONS

OF STEROID

CONCENTRATIONS

IN

Mean calculated concentrations (pg/2 ml) using (b) Peak heights

(a) Integrated areas

C.V.

2.07 2.Rl 2.75 0.99 3.49 1.45 1.10

9.1 4.8 11.6 7.2 a.4 5.4 5.2

C.V. (%a)

(%o) 1.91 2.41 2.29 1.14 2.90 1.49 1.24

9.6 4.4 9.9 3.9 5.3 3.7 6.8

The automatic injection and calculation of results led to the need for preliminary extraction and silylation to be as simple and rapid as possible. Bacterial fl-glucuronidase from E. coli permitted hydrolysis within 1 h. Unlike a more polar solvent such as ethyl acetate, chloroform produced extracts sufficiently clean for direct conversion to TMS derivatives. To improve the recovery of the polar cortisol metabolites, particularly cortol, but also THF and cortolone, Na,SO, was added to the urine before extraction (Table VII) and very small volumes of aqueous wash were used. With this procedure only a single derivatisation step was necessary. Different methods and reagents can be used to form trimethylsilylated steroids [ 91. A double derivative is often used for capillary column GLC of urine extracts; 0x0 groups at C20 and Cl7 are converted to methoximes before TMS formation [l-5]. However, the stabilisation of the reactive dihydroxy acetone side chain of corticosteroids such as THE, THF and THS can be achieved using a single base-catalysed silylation leading to a TMS-enol in the side chain [lo]. The successful formation of these TMS-enol derivatives requires a dry extract free of traces of acid and therefore no aqueous washes containing acetic acid were used at the extraction stage. TABLE VII EFFECT OF Na2S04 Steroid

A E DHA 1lOHE PD PT THE THF Cortolone

ON THE RECOVERY

OF STEROIDS

FROM URINE

Recoveries * of 2 pg amounts of steroid from 2 ml urine (%) -- .-. Without Na2S04

With Na2.904

104 97 104 106 104 110 98 86 77 36

100 93 103 95 96 100 103 107 al3 71

* Mean of duplicate determinations.

248

Table VIII shows the mean excretion and ranges for seven steroid metabolites in healthy men and women measured by different workers using glass capillary columns. Our mean 24-h excretion of androsterone and aetiocholanolone by 8 female subjects was lower than in 8 male subjects, although there was considerable overlap of the range for aetiocholanolone in the two groups. Bailey et al. [5] observed a higher mean excretion of androsterone and aetiocholanolone in females and a lower excretion of androsterone in males compared with the present study. Ros and Sommerville [2] claimed a higher mean excretion for androsterone than aetiocholanolone by 12 healthy women but their capillary column traces show aetiocholanolone to be the higher of the two. Mean excretion of pregnanediol and pregnanetriol by women during the follicular phase of the menstrual cycle and of pregnanediol by men was found to be less than 1 mg by all workers. However, we found the mean excretion of pregnanetriol by men to be twice the mean found by Bailey et al. [5] but in good agreement with levels measured by Vollmin [ 11. Mean excretion of THE and THF was about 50% lower in females than males, with THE always the major corticosteroid excreted in both groups. Ros et al. [2] failed to detect either of these principal corticosteroid metabolites. Their method for derivative formation appeared to be unsuitable for urinary extracts of these steroids with a dihydroxyacetone side chain. Viillmin [l] found 7.2 and 4.6 mg of THE in two males but did not quantitate THF which was barely separated from allo THF. Mean excretion of cortolone was also lower in females than males. Ros and Sommerville [2] found a similar mean in their study of female subjects but with a very wide range of excretion. The higher mean obtained for males is compatible with the greater excretion of THE and THF in males than females. During the luteal phase of the menstrual cycle three subjects excreted 2.0, 2.4 and 3.5 mg of pregnanediol. Bailey et al. [5] found a mean of 2.5 mg similar to 2.4 mg found by Ros and Sommerville [2]. In agreement with these workers, we found pregnanetriol in higher concentration during the luteal phase. Levels of 1.2, 1.0 and 1.0 mg in our study are supported by a mean of 0.92 mg found by Bailey et al. [5] and 0.87 mg by Ros and Sommerville [2]. We found excretion of the five other metabolites quantitated to be within the ranges measured during the follicular phase. For capillary column methods many workers use Helix pomatia enzymepreparations containing sulphatase as well as P-glucuronidase activity. However, of the two principal urinary steroid sulphates androsterone and DHA, the former is not hydrolysed by molluscan sulphatase (Leon et al. [ll]). This unhydrolysed fraction represents about 20% of androsterone excretion. Although 70% of DHA is excreted as a sulphate conjugate it is only a minor component of androgen excretion. Bailey et al. [5] found a mean of 0.4 and 0.3 mg/24 h in normal males and females, respectively, while Ros and Sommerville [2] observed a mean of 0.2 mg/24 h in normal females. In the region where 11-hydroxylated derivatives of androsterone and aetiocholanolone were eluted several small poorly resolved peaks were obtained and we were unable to identify and quantitate these steroids. Moreover, pregnanolone and 11/3-hydroxyaetiocholanolone occurring in similar low concentration in urine were not separated on our 20-m column. Bailey et al. [5] used bistri-

STEROID

EXCRETION

BY NORMAL

12

8

0.46 (0.18-0.73)

Not detected

Not detected

0.54 (0.06-1.49)

Cortolone 0.70 (0.61-0.89)

THF 1.1 (0.93-1.7)

2.05 (1.02-3.64)

0.65 (0.33-1.14)

THE 2.1 (1.0-3.1)

2.72 (1.06-4.94)

12

2.36 (1.19-4.13)

0.37 (0.15-Q.70)

PD

-

1.80 (0.98-2.67)

20

1.6 (0.84-2.3)

E

-

1.2 (0.61-1.8)

8

A PT

-

0.38 (0.19-0.56)

0.49 (0.26-0.70)

0.66 (0.49--0.95)

-

-

2

8

2

12

8

No. of subjects

No. of subjects

Steroid

Males

h.

SUBYECTS

Females

Mean and range (in parentheses) presented as mg/24

URINARY

TABLE VIII

-

-

6.89 (4.60-7.17)

-

2.2 (1.1-3.0)

4.8 (2.9-6.2)

1.2 (0.9-1.6)

Cortolone

THF

THE

0.71 (0.49--0.93)

-

0.43 (0.28-0.63)

0.44 (0.17-0.80)

PD

2.25 (1.82-2.67)

-

2.52 (1.17-4.83)

2.6 (1.5-3.5)

E

2.15 (1.68-2.61)

2.03 (1.06-3.22)

2.7 (1.94.2)

A

Steroids

1.55 (1.21-1.89)

-

0.67 (0.32-0.99)

1.4 [1.1---x.7)

PT

et al.

VoIlmin [l]

Ros and SommenriIle [ 21

Present series

VtiIImin [l]

Ros and Sommerville [23

151

Bailey

Present series

Reference

250

methylacet~ide and t~metl~ylchl~ros~ane conditions under which di-TMS derivatives of the ll-hydroxylated androgens are formed and thus pregnanolone was separated from 1 lfl-hydroxyandrosterone and 11/3-hydroxyaetiocholanolone. They found a mean of 0.27 mg for prenanolone and 0.41 mg for llflhydroxyaeti~~hol~olone in the follicular phase of the menstrual cycle and 0.52 mg and 0.26 mg, respectively, in the luteal phase, with a mean of 0.14 mg and 0.31 mg in healthy males, confirming the relatively minor importance of these metabolites. These workers found a mean excretion for llfl-hydroxyandrosterone to be 0.70, 0.59 and 0,50 mg in the follicular phase, luteal phase and in healthy males respectively, only slightly higher than the values found for all-hydroxyaetio~holanolone. Under the conditions used for derivative formation ll-oxoaetiocholanolone and ll-oxoandrosterone each gave rise to two derivatives due to partial enolisation of the 17-ketone group giving a mixture of mono and bis-TMS ethers [12]. This and the low levels of excretion made detection and quantitation difficult+ Bailey et al. [S] found a mean value of about 0.1 mg for ll-oxoandrosterone and about 0.5 mg for 11”oxoaetiocholanolone, The separation of pregnanetriolone from THS and the major corticosteroid metabolites permits the specific enzyme deficiency in congenital adrenal hyperplasia (CAH) to be determined. ~regnanetriolone not detectable in normals or ll-hydroxylase deficiency is produced when a 21-hydroxylase deficiency is present. ll-Hydroxylase deficiency is characterised by excretion of THS and also tetrahydro DOC (THDOC) in the hypertensive form of the disease. The latter steroid was not separated from pre~anetriolo~~e under the conditions used. Since pregnanetriolone is not produced in hypertensive CAH the presence of THDOC could be unambiguously established. Fig. 4 shows the urinary profile of a 5-year-old female patient with non-hypertensive 11-hydroxylase deficiency receiving inadequate suppression therapy. THS (1.7 mg) not detectable in normal urines was the principal urinary metabolite excreted. Two related metabolites, hexahydro S, 200 (130 pg) and 2Op (490 pg) previously reported in CAH by Halperin et al. [13] were also detected. In agreement with their findings, of the two isomers the 2Op was the major component. Of the urinary metabolites present in normal subjects, THE (200 erg), THF (40 @g) and cortolone (60 gg) were excreted in very low concentration. Androsterone, aetiocholanolone and pregnanetriol were not raised at concentrations of 90,80 and 80 Erg/24h, respectively, Although the chnical value of urinary steroid profiles is well established, qualitative investigations obtained on random urine samples have not previously been reported. Fig. 5 shows a profile obtained from a random urine sample from a healthy female. The large pregnanediol peak confirms that this sample was collected during the luteal phase of the menstrual cycle. The relative amounts of pregnanetriol, THE and THF are typical of normal cortisol production. Inefficient production of cortisol as in 21-hydroxyfase or ll-hydroxylase deficiency is characterised by a dominant peak for pregnanetriol or THS (Fig. 4) compared with either THE or THF. Overproduction of cortisol results in a much higher peak for THF compared with THE. This is shown in a urine profile from a 6%year-old women with an ACPH secreting tumour of the fung (Fig. 6) where THF was the major urine metaboiite produced. Cortol usually

251

60

45

30

15

min

40

Fig. 4. Gas chromatographic separation of urinary steroid TMS ethers from a 5-year-old girl on inadequate steroid therapy for congenital adrenal hyperplasia due to sn 11-hydroxylase deficiency.

not detectable was easily identified. The peak eluted closely after it is likely to be its 200 isomer. Further evidence of excess cortisol production is the presence of 11/3-hydroxylated aetiocholanolone and androsterone, metabolites of THF and its 5c~ isomer, respectively. Other abnormalities are the high proportion of aetiocholanolone compared with androsterone and detectable amounts ot THS. Pregnanediol and pregnanetriol were not raised. In neonates investigation of adrenal function from analysis of a random urine sample is particularly useful since 24-h samples can be difficult to collect. Thus a urine profile from a neonate in salt-losing crisis due to a 21-hydroxylase deficiency would have a major pregnanetriol peak. The presence of pregnanetiolone would confirm the diagnosis. Moreover, using the fast acting /3-glucuronidase preparation from E. coli, the urine could be analysed and the diagnosis established well within a working day. Although in most disorders of the adrenal, this urgency is not required, it is useful to have a method, the results of which are not dependent on the accuracy of the collection of the 24-h urine. We have found profiling of urinary steroids valuable for qualitative and quan-

252

60

A5

30

__________~

15

Nl‘lV

y

Fig. 5. Gas chromatographic separation of steroid TMS ethers from a random urine sample of a healthy female during the luteal phase of the menstrual cycle.

titative investigations. The use of the computing integrator has improved the accuracy of our results at higher concentrations as well as eliminating the need for time-~onsu~ng calculations. Some of the other facilities available on the integrator which were not used include the application of different baseline corrections to groups of fused peaks. A choice of horizontal or trapezoidal baseline corrections is possible. With the high-resolving capacity of the capillary column large groups of partially resolved peaks were not obtained for the steroids of major interest. Another option includes the automatic doubling of the peak width parameter to allow for broader peaks occurring during later stages of an analysis. With temperature programming peak broadening did not occur. A further option includes inhibiting integration in the middle of an analysis where components of interest are not being eluted. It is also possible to store a complete set of parameters and calibration factors for separate unrelated analyses; one in each of four memory files provided.

253

Fig. 6. Gas chromatographic separation of steroid TMS ethers from the urine of a female patient with an ACTH secreting tumour of the lung.

Acknowledgements This work was supported by a grant awarded to Professor C.H. Gray by the Cancer Research Campaign. References 1 2 3 4 5 6 7 8 9 10 11 12 13

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