Index

absolute configuration 3, 18, 20 accreditation 663 accuracy 438, 649 see also inaccuracy; bias

accurate mass 94 acetonides, derivatives for mass spectrometry 106

17-acetoxy-6a-methylpregn-4-ene-2,30-dione 14, 83, 88

17 fJ-acetoxyandrost-4-en-3-one 15 acid hydrolysis 429,447, 449

of steroid sulphates 122-124 ACTH test 232, 244, 249 adrenal function test 232 adrenocorticotrophin hormone see ACTH

adrenosterone 51 affini ty 444 affinity chromatography in C19 steroid isolation 321, 335

affinity column, use in extraction of vitamin D metabolites 574

agarose 448 agglutination inhibition 434 albumin 451,452 aldosterone 7, 11, 660, 688, 689, 691

assays for 250-252 excretion rate, measurement of urinary

metabolites 252 18-glucuronide assay 252 synthesis from cholesterol 230

alkaline phosphatase 439 alkane boronates, derivatives for mass spectrometry 106

alkenes 18, 23, 46, 47, 61, 84 alkynes 46, 47, 52, 61, 84 All-Laboratory Trimmed Mean 667, 671,

672,673 allocholane 4 allopregnane 4 allotetrahydrocortisol 690 ALTM see All-Laboratory Trimmed

Mean aluminium oxide, in column chromatography 131-132

amberlite XAD resins 528, 529, 539 amniotic fluid, cortisol in 242 anabolic steroids

biological fluids for analysis of 493

conjugates, hydrolysis of 489--491 derivatives of, and GC-MS 498-509,

511 extraction and purification of 485--492

HPLC of 144 immunoassays, anabolic steroids for

493--497 immunochromatography of 489 in food, extraction of 120 liquid-phase extraction of 487--488 measurement by GC-MS 165 metabolism of 475--485 mixed-phase extraction 488--489 natural hormone administration,

detection of 509-518 preparation of 475 screening analysis of 492-503 solid-phase extraction of 486--487 structural requirements for 474--475 synthetic, confirmatory analysis of

503-509 TLC of 489 trivial, systematic and trade names of 469

undesirable effects of 473--474 use and abuse 468--472 see also individual steroids

analysis, steps in 115 et seq. analytical goals 656, 657 androgen, biosynthetic pathway 230 androgen glucuronides, HPLC-MS of

176 androgen sulphate 446 androgens

biosynthesis of 268-269 characterisation of 280-291, 294-317,

324-332 by GC profiling 301-303 by HPLC 295-301 by MS 305-318 by specific radioactivity measurements 294-295

detection and quantification by UV, NMR 273-274

extraction of 276-279, 280-291, 292, 324-332 by liquid-phase 277-279, 292 by solid-phase 276-277

698

androgens (contd) immunoassays (lAs) for 323-353

chemiluminescent assays 343-346 ELISAs and Els 340-343 f1uorometric assays 346-349 problems with direct methods 336-337

radioimmunoassays 349-353 sample preparation in 334-335

measurement in plasma by GC-MS 164

metabolism of 269-272 nature and sources of 268 physiological actions of 273 purification of 280-291, 292-294,

324-332 by C18 Sep-Pak cartridges 292-293 by Lipidex 293 triethylaminohydroxypropyl (TEAP)-LH20 292-294

separation by TLC 275-276,280-291, 324-332

androst-4-en-19-al-3,17-dione 53 androst-5-ene-3a, 17 fi-diol 17 -acetate 15 androst-5-ene-3fi, 17 fi-diol 17-benzoate

51 androst-5-ene-3a, 17,B-diyl acetate 15 androstane 2, 4, 12 5a-androstane-3a 17 fi-diol 13 5a-androstane-3a,16a-17 fi-triol, characterisation of by GC-MS in Sertoli cells 313

androstanediols derivatives for, in GC-MS 500, 507 formation from testosterone 477 fragmentation of, in GC-MS 505 mass spectra of 503, 506, 508 selected ions for, in GC-MS 501 separation of, from boldenone 508

5a-androstanediols formation of 269-271, 313 glucuronidation of 271 measurement of by GC-MS 280-291,

307, 311-312 RIAs for glucuronides of 324-332,

352-353 5-androstene-5fi,17 fi-diol

assays for 280-291, 324-332 biosynthesis of 269 3-sulphate of RIA for 353 sulpho-conjugation of 352

androstenedione 6, 86, 437, 660, 688, 691 assays for 246-247

4-androstenedione androgenicity of 273 as internal standard 310 assay of by GC-MS 280-291, 314 assay of by lAs 324-332, 342-343

INDEX

formation of 268-269 hydroxy derivatives HPLC of 297 metabolism of 269-270

16-androstenes, measurement by GC-MS 164

androsterone 11, 660, 690 anisotropic effects in NMR spectroscopy 62

anti-estrogens 441 antibodies 433 antibody 652, 658 antigen 439 antigenic protein 436 aromatic steroids 5, 11, 12, 14, 35, 47, 53,

61, 62, 79, 80, 84 ascorbic acid 449 assisted conception 649 AS TED, automated system for cortisol

analysis 239 audit 662 automated immunoanalyser 439, 456 avidity 441 axial bonds 9, 63

Baljet reaction 625 BCR see Community Bureau of Reference

beads, coated 439 benzo-15-crown-5 ether, in LC-MS of vitamin D metabolites 584

Bergmeyer Conferences 650 bias 649, 659, 670 see also inaccuracy bile acid

antiserum 550 breath test 552 glucuronides 527, 528, 545, 547-549 nomenclature 528 sulphates 539, 545-547

bile acid derivatives l-anthroyl nitrite 536 bromoacetylpyrene 536, 540 bromoethyl-7-methoxy coumarin 536 dansylhydrazones 536 I -125 histamine 549 1-125 tyramine 549 methyl esters 545, 548 oximes 537 pre-column 536 trifluoroacetate 548 trimethylsilyl ethers 546-548

bile acid esters, use in HPLC 157 bile acid HPLC, detection by use of 3c£­hydroxysteroid dehydrogenase 154-155, 157

bile acid metabolism biosynthesis 527 deconjugation 527 enterohepatic circulation 527, 528

inborn errors 551 primary 527 secondary 527

bile acid methods affinity chromatography 550 breath test 552 enzymatic assay 531-535 enzyme electrode 530 extraction 529 gas-liquid chromatography 545-549 high performance liquid

chromatography 535-545 hydrolysis 546, 548 immunoassay 549-551 mass spectrometry 551-552 SeHCAT 552 separation 530, 535 solvolysis 546, 548

bile acids 8, 11, 68 chenodeoxycholic 527, 528, 549 cholic 527, 528, 549 deoxycholic 527, 528 detection after HPLC 154-155 7-,12-dihydroxy-5-cholanic 538 glycine-conjugated 527, 528, 536, 538 HPLC of 144, 145, 151 hydrolysis of conjugates 124 3,B-hydroxy-5-cholenic acid 530 1-P-hydroxycholic 549 in bile 532-533, 540--545 in faeces 529, 533-534, 552 in serum 529, 532-533, 552 in urine 528, 552 lithocholic 527, 530, 538, 539, 549 ,B-muricholic 549 23-selenium-25-homotaurocholic

[SeHCA T] 552 taurine-conjugated 527, 528, 538 unconjugated 536, 538, 541 ursodeoxycholic 538, 539, 549

binding proteins 650 bioassay 432, 457 biological variation 657, 662 biologically active estrogen 443, 452 bioluminescent assay 439 blank values 440, 445 blood spot, assay for 17-hydroxyprogesterone 244

Blood Transfusion Service 658, 665, 684 blunders 684 boldenone

conjugation of 485 mass spectrum (MO-TMS) of 507 metabolism of, in equine and human

478-479,485 separation from androstanediol 508 solid-phase extraction of 486 structure of 471

INDEX 699

boldenone sulphate, HPLC-MS of 181 Bond-Elut 572-573

C1s-OH cartridges, for extraction and separation 575

C1s-OH, in vitamin D metabolite extraction 572-573

C18, use in extraction of vitamin D metabolites 598

bone gla protein see osteocalcin 566 bovine serum albumin see BSA box and whisker plots 671, 676 brassinolide 635 brassinosteroids 635-637 British Laboratory Ware Association

656 broad band decoupling in 13C NMR spectra 85

BSA antigenic protein 448 methylated 446

bufodienolide, definition 621 bufotoxin 621, 622 butylboronate, formation of with vitamin D metabolites prior to GC-MS 596

t-butyldimethylsilyl ethers, derivatives for mass spectrometry 106

t-butyldimethylsilyl oxime, derivatives for mass spectrometry 107

13C NMR spectroscopy 84 C19 steroid conjugates

chemiluminescent assay for 345 formation of 271-272, 319 hydrolysis of 318-319 immunoassays for 341 RIAs for 352-353 separation of by FAB 320--323 separation of by HPLC 280--291, 319-320, 324-332

CAH need for extraction in assay for 17-hydroxyprogesterone 121-122

use of column chromatography 131 calbindin-9K gene 567 calcidiol 20 see also 25-hydroxyvitamin

D3 calciferol BP 562 calciol 20 calcipotriol (MC903), structure 568 calcipotriol

assay of in human plasma 593 NMR of metabolite of 608 structure 608 therapeutic effects 570

calcitetrol 20 calcitriol 20 see also 1,25-dihydroxyvitamin D3

calcitroic acid, structure 568

700

calf thymus, use in vitamin D metabolite assay 589

calibrated standards, availability of 234 calibration 441, 650 campestane 22 CAP see College of American

Pathologists carbonyl compounds 33, 49, 50, 54 carboxylic acids 16, 55 cardenolide, definition 621 cardiotonic steroids 621-626 catechol estrogens 448, 454

pregnancy urine 449 RIA 449

catechol-O-methyl transferase 449 CBG see corticosteroid binding globulin celite 430, 431

chromatography 442 columns 442,451

centrifugal ultrafiltration 338 dialysis 394-395 testosterone assay by 339

cerebroteninous xanthomotosis, use of GC-MS 166

Certified Reference Materials 679-680, 681 see also Community Bureau of Reference

chair conformation 9 charcoal 440, 442 chemical analogues of vitamin D 569 chemical ionisation (CI) 94, 95, 158-159

in mass spectrometry 94-95, 97 chemical shift in NMR spectroscopy 59 chemiluminescence 439

as enzymeimmunoassay endpoint 389-390

assay of pregnanediol 3o:-glucuronide in urine 401

assay of progesterone 390 chemiluminescent immunoassays 343-346

commercially available kits for 324-332

isoluminol derivatives in 344-345 testosterone glucuronide measurement by 345-346

testosterone measurement by 346 chenodeoxycholic acid 8, 11 chiroptical properties 36 chloramine T 443 cholane 2, 3, 4 cholecalciferol 8, 20 see vitamin D3 cholestane 2, 4 cholest-4-en-3-one 13 cholest-5-en-3fJ-ol 8, 11 cholest-5-ene 12 cholesterol 8, 11, 86, 96, 371

conversion into steroid hormones 230 EI mass spectrum of 96

INDEX

GC-MS for validating assay of 166 in food, extraction of 120 sulphate, HPLC-MS of 179

cholic acid 8, 11 cholylglycine hydrolase 546, 548 chromatography 442, 446

partition 443 prior to immunoassay 234-237

CIA see chemiluminescent immunoassay circular dichroism 36 CLlA see Clinical Laboratory

Improvement Act Clin-Elut 573 Clinical Laboratory Improvement Act 691

Clinical Pathology Accreditation (UK) Ltd 663

closed systems see automated immunoanalysers

CN cartridges, for separation of oxo­steroids 577

collagen type-I gene 567 College of American Pathologists 664, 691

collisionally activated decomposition, in mass spectrometry 94

colorimetry 432, 433 column chromatography 129-135,444, 457

adsorption 131 effect of packing on resolution 130 review of 131 separation of androstanedione and androstenedione 130

column packing material for HPLC 142-152

column switching, during HPLC 157-158

columns used for GLC 138 Community Bureau of Reference 663,

679 see also Certified Reference Materials

commutability 666 competitive assays 433 competitive binding assay 442 competitive protein binding 374-375,408, 409 see also CPB

configuration 3, 18, 20 confirmatory analysis

by MS-MS 509 criteria for 503-505 of methenolone 504-505 of natural anabolic steroids 492,

509-518 of synthetic anabolic steroids 503-509

conformation 8, 10, 33 congenital adrenal hyperplasia (CAH) 231-232, 246, 251, 257 see also CAH

conjugated dienes 26 estrogens 428 trienes 26 lX,fJ-unsaturated ketones 26

conjugates 5, 15 Conn's syndrome, steroids assays in 254 Continuous Quality Improvement 648 contour plots in 1 H NMR spectroscopy

88 control rules 661 coprostane 4 cortical hormones 7, 11 corticosteroid binding globulin 375, 379,

391 corticosteroid glucuronides, HPLC~MS

of 177~179

corticosteroids definition 229 HPLC of 145 measurement in plasma by GC~MS 163~165

separation by aluminium oxide chromatography 132

synthetic 243 corticosterone 7, 11, 86

assays for 257~258

HPLC of 144 metabolites in urine 257

cortisol 7, 11, 86 analytical goals 657 Certified Reference Materials 679~680,

681 EQA serum concentration 666, 668 estimation of 237~242

external quality assurance of assays 238 GC~MS for validating assay of 166 HPLC of 145, 146 HPLC~MS of 170-172 in amniotic fluid 242 in saliva 237 in urine 241 IQC materials 660 measurement

by fluorimetry or colorimetry 232 by GC~MS 163 by polarisation immunoassay 240 by scintillation proximity assay 240

methods 651 specificity of immunoassay of 240 standardisation 650 synthesis from cholesterol 230 UK NEQAS GC~MS targeting exercise 682, 683, 684-685

UK NEQAS participation 654 UK NEQAS performance 671, 674,

675, 676

INDEX

urinary free 685, 660 WHO EQAS 692

cortisone 7, 11 21-acetate 71, 90 HPLC of 146 measurement by GC~MS 163

cortoic acids HPLC of 146 HPLC~MS of 175~176

fJ-cortolic acid, HPLC~MS of 175

701

fJ-cortolonic acid, HPLC~MS of 175 cortolonoic acids, HPLC of 146 COSY NMR spectra 87 counter-current distribution 430, 431 CPA (UK) Ltd see Clinical Pathology Accreditation (UK) Ltd

CPB 433, 446 see also competitive protein binding

CQI see Continuous Quality Improvement

CRM see Certified Reference Materials cross-reaction 436, 450, 451

synergism 438 cross-reactivity

of testosterone and 51X-DHT, in immunoassays 340-341

of testosterone and 51X-DHT, in RIAs 351

with commercial immunoassay kits 233

CUSUM 661 cutaneous androgen metabolism, markers for 269~270, 272

cyanogen bromide, coupling of antisera 387

cyc10dextrin derivatives with vitamin D metabolites 609~61O

in steroid extraction 128 in steroid HPLC 143

cyc10dextrins in HPLC 152~154

in HPLC mobile phase 579 cytochrome P450

in 11X-hydroxylation 566 in 25-hydroxylation 565

cytoreceptor assay, in measurement of 1,25-dihydroxyvitamin D 590

cytosol 442 cytosol sheep uterus 444 cytosol-target tissue 441

Daivonex see calcipotriol Danazol 441 data 661, 667 daturametelin-F 628, 629 DBP (vitamin D binding protein) 564

antibody to 574

702

DBP (vitamin D binding protein) (contd) as binding protein in saturation analysis

of vitamin D metabolites 588 binding affinities of vitamin D3 564 precipitation with ammonium sulphate 564

use in extraction of 1,25-dihydroxyvitamin D 574

use in extraction of metabolites from plasma 564

dehydrated vitamin D metabolites, use in GC~MS 601

7-dehydrocholesterol 562 measurement in skin 586 UV spectrum 564

dehydroepiandrosterone 6, 11, 660, 690 see also DHA

dehydroepiandrosterone (DHA) biosynthesis of 268~269

GC profiling of 301~303 immunoassays for 324~332

metabolism of in equine testis 314-315 separation of by HPLC 295~300 sulphoconjugation of 273~274

dehydroepiandrosterone (DHEA), effect of enzyme hydrolysis 124

dehydroepiandrosterone sulphate 660, 688, 689, 691 assays for 248~249

HPLC~MS of 180 delayed fluorescence assay 433 21-deoxyaldosterone, assays for 249~250 deoxycholic acid 8, 11 deoxycorticosterone 7, 11 ll-deoxycorticosterone, assays for 258 21-deoxycorticosterone, assays for 249~250

ll-deoxycortisol 660, 691 assays for 245~246

HPLC of 145 21-deoxycortisol, assays for 249~250

I1-deoxytetrahydrocortisol 690 Department of Health 655 DEPT NMS spectra 85 derivatisation

for mass spectrometry 94, 95, 105 in mass spectrometry 94, 105

deuteriated steroids, as internal standards 116

deuterium labelled internal standards 575 deuterium NMR spectroscopy 91 deuterium-proton exchange 80 Deutsche Gesellschaft fUr Klinische Chemie 690

dexamethasone assay of 243 determination by HPLC 157 suppression test 232

INDEX

dextran coated charcoal 433, 437, 443 see also charcoal

DGKC see Deutsche Gesellschaft fUr Klinische Chemie

DHA sulphate 446 assay of by GC~FAB 280~291,

320-323 formation of 271 hydrolysis of 318~319 immunoassays for 335~336, 343 occurrence in urine 272 RIAs for 324-332 separation of by HPLC 319~320

separation of by TLC 319 DHEA see dehydroepiandrosterone DHEAS see dehydroepiandrosterone sulphate

50:-DHT 441 dialysis see centrifugal ultrafiltration­dialysis; equilibrium dialysis; rate dialysis

dianabol, determination by HPLC 157 diethyl ether 428, 443 diethyl stilbestrol 5, 441 differential ammonium sulphate technique, testosterone assay by 339

digitalis steroids 621 ~626 digoxin 621, 622, 625, 626

HPLC of 144 dihydroequilenin-17 0: 451 dihydroequilenin-17 {3 451 170:-dihydroequilenin sulphate 454 dihydroprogesterone 408-409

in urine 398~399

dihydrotachysterol GC~MS in study of metabolism 602~604

HPLC of metabolites 582 HPLC separation of metabolites 582 UV spectrum 582

dihydrotachysterol3 , structure of 569 dihydrotesterone 6

detection of 515~516

effects on TIE ratio of 516 50:-dihydrotestosterone see 50:-DHT 50:-dihydrotestosterone (50:-DHT)

19-norderivative as internal standard 310

cross-reaction of with testosterone in ELISA 340-341, 351

formation from testosterone 299 glucuronidation of 270 measurement of by GC~MS 280-291 metabolism of 271

22,23-dihydroxyergosterol 17 10:, 25-dihydroxycholeca1ciferol 20 30:, 70:-dihydroxy-5{3-cholan-24-oic acid

11

3tX, 7 P-dihydroxy-5p-cholan-24-oic acid 11

(24R)-24, 25-dihydroxycholesterol 17 1,25-dihydroxy-16-ene-vitamin D 3, structure of 569

1 tX,25-dih ydroxydih ydrotachysterol mass spectrum of per-trimethylsilyl

ether 603 structure 603

lIP, 21-dihydroxypregn-4-ene-3,20-dione 7,11,86

17,21-dihydroxypregn-4-ene-3,11,20-trione 7, 11

15P,17tX-dihydroxypregnanolone, in urine as marker for 21-hydroxylase deficiency 245

1,25-dihydroxyvitamin D measurement by cytoreceptor assay

590 measurement by GC-MS 596-597

24,25-dihydroxyvitamin D, measurement by GC-MS 596-597

25,26-dihydroxyvitamin D, measurement by GC-MS 596-597

1,25-dihydroxyvitamin D3 24-hydroxylation of 567 26-hydroxylation of 567 binding to the VDR 566 catabolism of 567-569 control of production 566 in cell differentiation 567 in tumour regression 567 measurement by scintillation proximity

assay 591 metabolites of 568-569 molecular events in target cells

566-567 physiological effects 566 receptors in skin 567 stimulation of production of

osteocalcin 566 structure 568 26,23-lactone, structure 568

24,25-dihydroxyvitamin D3 567-568 structure 568

25,26-dihydroxyvitamin D 3, structure 568 dimethyldichlorosilane, used to treat glass

surfaces 118 23,24-dinor-5p-cholan-22-oic acid 19 direct assay 438, 447 direct systems 441 dissociation-enhanced lanthanides, f1uoroimmunoassay (DELFIA) 348

double-antibody separation 437 double-isotope derivatisation 433 drift 659

EC see European Union

INDEX

ecdysone 631 ecdysteroids 631-634 ecdysterone 631, 632

703

EIA see enzyme immunoassay electrochemical detectors for HPLC 154 electron 94, 95

impact, in mass spectrometry 94, 96 ionisation (EI), in mass spectrometry

94, 95, 158 et seq. electron capture 94, 95

in mass spectrometry 94--95, 97 electrospray 94, 95 electrospray MS 166 et seq. electrospray-mass spectrometry 94-95, 97 ELISA 439 see also enzyme-linked immunosorbent assays

end-capping of microparticulate silica 572,575

endogenous binding proteins interference 441

enol ether, derivative for mass spectrometry 107

enzyme hydrolysis 446,447,451 of steroid glucuronides 122-124

enzyme immunoassay 385-389,401-402, 415

for 25-hydroxyvitamin D3 574 enzyme immunoassays 324--332, 340-343

4-androstenedione 342-343 for DHA sulphate 343 for testosterone 340-342

enzyme labelling 433 enzyme-linked immunosorbent assays

324--332, 340-343 C19 steroid cross-reactivities in

341-342 effect of hapten heterology on 341-342 for 4-androstenedione 342-343 for testosterone 340-342 principles of 340 structure of haptens in 340-341 see also ELISA

enzymes in steroid synthesis 230 16-epi-estriol 431 epimerisation, mechanism of 482-483 epitestosterone

excretion of 511 testicular production of 510 see also hormone ratios

epitopes 433 11 p,18-epoxy-18-hydroxypregn-4-ene-3,20-dione 11

EQA see External Quality Assessment EQASs see External Quality Assessment, Schemes

equatorial bonds 9, 63 equilenin 5, 450, 451, 454 equilenin sulphate 450

704

equilibrium dialysis (EQD) 392-393, 452 principles of 338 testosterone measurement by 338

equilin 450 3H-equilin 451 equilin 3-hemisuccinate-BSA 451 equilin 3-sulphate 451 equilin sulphate 450 equilin-17 -( O-carboxy-methyl)-oxime-BSA 450

equilin-17 -( O-carboxymethyl)-oxime-[125IJiodohistamine 450

equine estrogens 441 ergocalciferol 8, 20 see vitamin D2 ergosta-5,7-dien-3tJ-ol 17 (22E)-ergosta-5, 7, 22-trien-3tJ-ol 17 ergostane 22 ergosterol 17 error 656, 657 erythrocyte 449 esters 14, 50, 56 estetrol 453, 454 estradiol 5, II, 428, 431, 435, 436, 437, 439,440,441,442,443,446,447,452,453, 454

-17a 435,441,454 -17 tJ see estradiol analytical goals 657 CAP PT scheme 691 -6-( O-carboxymethyl)-oxime-BSA 443, 444, 445

-6-( O-carboxymethyl)-oxime-(2-[125IJ)iodohistamine 443

conjugates 439 EQA serum concentration 666, 668 extraction using affinity column 574 fatty acid esters, measurement by GC-MS 165

-17 fatty esters 456 glucuronide, urine 429 17tJ-glucuronoside 447,448 3-glucuronoside 447, 448 3-hemisuccinate-BSA 436 -lla-hemisuccinate-BSA 436 -17 tJ hemisuccinate-BSA 436, 444, 445 HPLC-MS of fatty esters 172-173 2-hydroxylase 432 4-hydroxylase 432 IQC materials 660 Murex EQA scheme 689 oral HRT effects on results 685, 686, 687

reduction 445 RIA 454 saliva 443 -17a serum 444 serum concentration 444, 649

men 442

INDEX

postmenopausal women 442, 443 sulphate 452 -17 tJ sulphate 455 UK NEQAS participation 654 UK NEQAS performance 671, 672, 674, 675, 676

UK NEQAS recovery experiment 678, 679

urine 442 WHO NEQAS 692

17 tJ-estradiol see estradiol 17 tJ-estradiol-6-(C-carboxymethyl)-oxime-BSA 435

3H-estradiol 442, 444, 445 estrane 2,4 estranediols

formation of, from nandrolone 477, 516-517

GC-MS fragmentation of 505 selected ions for, in GC-MS 501 structures of 477 urinary excretion of 517

estra-5(1O)-ene 12 estra-l,3,5(1O)-triene 12 estra-l,3,5(1O)-triene-3,17a-diol 11 estra-l,3,5(1O)-triene-3,17 tJ-diol 5, 11 estra-l,3,5(1O)-triene-3,16a, 17tJ-triol 5, 11 5( 10)-estrenediol

in eq uine testis 517 selected ions for in GC-MS 501 structure of 516 use of, in nandrolone administration 517-518

estriol 5,86,428,431,441,446,447,448, 660,691 -3-(O-carboxymethyl)-oxime-BSA 445 -6-( O-carboxymethyl)-oxime-BSA 445, 446

glucuronide, urine 429 3-glucuronide 445, 455 16-glucuronide 455 17 -glucuronide 455 16a-glucuronide, urine 429 3-glucuronoside 447, 448 16a-glucuronoside 447,448 16tJ-glucuronoside 447 16-glucuronoside urine, non-pregnant women 448

saliva 453, 454 serum 445 3-sulphate 445 3,16,17-trihemisuccinate-BSA 451

3H-estriol 446, 447 3H-estriol 16-glucuronoside 448 estrogen glucuronide

plasma 447 urine 447

estrogen glucuronoside 452

3H-estrogen 447 estrogen-protein conjugates 434 estrogens 432, 660

extraction of esters 120 GLC amniotic fluid 432 GLC blood 432 GLC urine 432 HPLC of 148-150 separation by solvent extraction 121

estrone 5, 86, 428,431, 435, 436, 437, 439, 443,444,445,446,447,448,450,453,454

acetate 53 -3-( O-carboxymethyl)-oxime-BSA 446 -6-( O-carboxymethyl)-oxime-BSA 445 conjugates 439 3-glucosiduronate-bovine thyroglobulin 446

3-glucuronide 692 glucuronide amniotic fluid 429 glucuronide urine 429 3-glucuronoside 447, 448 3-hemisuccinate-BSA 437 3-methylphosphothionate 446 3-sulphate 451 3-sulphate 60(-hemisuccinate-BSA 446

3H-estrone 445 estrone sulphate 446, 452, 454

amniotic fluid 429 cow's milk 446 extraction ether-ethanol 430 plasma 446 serum 446 sow serum 446 urine 446

[3HJ-estrone 446 sulphate 446

ethers 14 ethinyl estradiol 441, 451, 453, 454 ethinylestradiol 3-methyl ether 451 ethisterone 52 ethynylestradiol 19 170(-ethynyl-17 -hydroxyestr-4-en-3-one 19 170(-ethynylestra-1, 3, 5(1O)-triene-3, 17 p-diol 19

etiocholanone 690 EU see European Union European Commission see European Union

European Union 656,663,693 exocyclic alkenes 32 External Quality Assessment 662-693

data processing 667, 670 definition 662 performance evaluation 667, 671-685 principles and practice 664--670 samples 665-666 Schemes 662, 689-692 survey method 664

INDEX

target values 666-667 see also UK NEQAS

extraction

705

selectivity by choice of solvents 121 use of ion exchange columns 134-135

extraction by solid phase material 124 et seq.

extraction of anabolic steroids by immunoaffinity chromatography 489

by liquid-phase 487-489 by solid-phase 486-489 by thin-layer chromatography 489

extraction of estrogens 428 extraction of steroids 117 et seq.

use of ammonium sulphate 118 use of solvents 118 et seq.

extraction of vitamin D 571-575 extraction of vitamin D metabolites

from plasma, use of DBP 564 prior to GC-MS 596, 598

Extrelut 573 E/Z-convention for alkenes 23

F AB/LSIMS 95 fast atom bombardment (F AB) 94 fast atom bombardment (F AB), mass spectrometry 94--95, 97

fatty acid esters 440 of steroids, measurement by GC-MS

165 fatty acids, removal from vitamin D extracts 571

FDA see Food & Drug Administration Federal Medical Society of Germany 689-690

feto-placental function 447 FIA see fluoroimmunoassay Florisil, in column chromatography 131 fluorescence 35

detection during HPLC 154 fluorimetric detection 453 fluorimetry 432, 433 fluoro-steroids 92 fluoroimmunoassays (FlAs) 346-349

commercially available kits for 324-332, 349

DELFIA enhancement in 348 europium 3H-liposome encapsulation in 349

17 P-hydroxysteroid dehydrogenase measurement by 348-349

lanthanide chelates in 348-349 linkage of steroids to fluorescent labels in 348-349

principles of 346-347 testosterone measurement by 347-348

follicular function 448

706

Food & Drug Administration 656 food steroids, extraction of 120 Fourier transform

IR spectroscopy 43 NMR spectroscopy 58

free progesterone determination by centrifugal ultrafiltration-dialysis 394-395

by equilibrium dialysis 392-393 by rate dialysis 394 by steady state gel filtration 393-394 by ultracentrifugation 394

free progesterone equivalence to saliva concentration 396

free unbound plasma steroid 566 functional group nomenclature 5, 13

gas chromatography coupling of with mass spectrometry

305-316 derivatives used in 303-304 separation of C19 steroids by 301-303 see also GLC

gas chromatography-mass spectrometry 94-95, 100, 103, 108,234 derivation of target values 666-667 derivatives of anabolic steroids for 498-503

full scan spectra of anabolic steroids 505-509

selected ions of anabolic steroids 499, 501

UK NEQAS studies 678-685, 682, 687 use in German EQA schemes 690 use in standardisation 650 see also GC-MS

gas liquid chromatography see GLC GC-MS 94,161-166,433,438,455,457

for hexafluoro-l,25-dih ydroxyvitamin D3 592

measurement of dehydrated vitamin D metabolites 601

of cortisol and 18-hydroxycortisol in urine 255

of medrogestone 409--410 of medroxyprogesterone acetate 411--412

of norethisterone 413--414 of pregnanediol 417 of progesterone 404--405 use for obtaining target values of vitamin D metabolites 600

use in determination of vitamin D metabolite structure 602 et seq.

use in studies of vitamin D metabolising enzymes 599

use of double focussing instrument in vitamin D measurement 601

INDEX

see also gas chromatography-mass spectrometry

gel filtration 428, 452 glass surfaces

binding of steroids to 118 destruction of steroids on 116

glassware, silanisation to prevent adsorption 573

GLC 137-141, 373-374, 401, 405, 432--433

capillary 433 derivatisation 433 derivatives used in 140-141 electron capture 433 flame ionisation 433 for aldosterone 252 methods of detection 141 of vitamin D metabolites 595 et seq. urinary steroid profile in normal man

142 use in assay of 18-hydroxycorticosterone 253

GLC-MS of vitamin D metabolites 594 et seq.

y-globulin sheep anti-rabbit 443 GLP see Good Laboratory Practice glucocorticoid, biosynthetic pathway 230 glucuronic acid 448

conjugation 449 fJ-glucuronidases 547 glucuronide 437, 439 17 fJ-glucuronides hydrolysis of 489--492 glycyrrhetinic acid, HPLC-MS of 174 glycyrrhizic acid, HPLC-MS of 174 GMP see Good Manufacturing Practice goat 443 gonadotrophins 434, 448 Good Laboratory Practice 648 Good Manufacturing Practice 648 graphitised carbon in steroid extraction

127-128

haemocyanin 436 hapten 435, 436, 442, 447 Helix pomatia, in steroid conjugate hydrolysis 124

hemiacetals 55 hepatitis Band C 658, 665 heteroannular dienes 30 heterodimers in activation of vitamin-D responsive genes 567

heteronuclear spin-spin coupling in NMR spectroscopy 86, 91, 92

hexafluoro-1,25-dihydroxyvitamin D 3,

measurement by GC-MS 165,592,596 high performance liquid chromatography

coupling with MS (thermospray) 316-317

separation of C19 steroid conjugates by 319-320

separation of C19 steroids by 295-301 high performance thin layer chromatography 577-578

high pressure liquid chromatography see HPLC

histamine 438 [125I]-histamine 445 HIV 658,665 homoannular dienes 30, 32 homodimers in activation of vitamin-D responsive genes 567

homonuclear spin-spin coupling in NMR spectroscopy 81

homosteroids 20 Hooker-Forbes test 372 hormone ratios

detection of testosterone administration and 510-515

dihydrotestosterone administration and 515-516

in biological outliers 514-515 isotope mass spectrometry and

513-514 testosterone/dehydroepiandrosterone and equine 518

testosterone/epitestosterone (T /E) 510-516

testosteronejluteinising hormone 510-513

hormone replacement therapy 655, 658, 685, 686, 687

24 hour urine collection, necessity for 232

HPLC 141-158,432,433,453,454,457 assessment of homogeneity of emerging peaks 579-581

automated 454 autosampling 454 box-car 454 catechol estrogens 453 choice of mobile phase 152-153 columns available 142-152 detection limit 454 detection of steroids 154-155 estrogens 454 identification of steroids 155-156 improved resolution of vitamin D2 and

tachysterolz 563-564 incorporation of cyclodextrins in mobile phase 579

ion-pair chromatography 153 linked to mass spectrometer 581 et seq. micellar chromatography 153 modern methodology 144-150 precolumn derivatisation 156--157 prior to immunoassay 235

INDEX 707

resolution of skin irradiation products 563

review of methods up to 1987 142 sample injection 153-154 separation of irradiation products of ergosterol 565

systems for vitamin D separation 578-579

use of cyano columns for isolation of vitamin D lactones 578

use of electrochemical methods for vitamin D metabolite analysis 586

use of photodiode array detection 579-583

see also high performance liquid chromatography

HPLC-MS 156 electrospray of vitamin D metabolites

585 of androgen glucuronides 176 of boldenone sulphate 181 of cholesterol sulphate 179 of corticosteroid glucuronides 177-179 of cortisol 170-172 of ,B-cortolic acid 175 of ,B-cortolonic acid 175 of dehydroepiandrosterone sulphate

180 of estradiol of fatty esters 172-173 of etrahydrocortisone-3-glucuronide

178 of glycyrrhetinic acid 174 of glycyrrhizic acid 174 of 4-hydroxyandrostene-3,17-dione 172 of 17-hydroxypregnenolone sulphate

182 of 5-hydroxyvitamin D3 172 of ortoic acids of 175-176 of 3-oxo-4-ene-steroids 169-172 of placental sulphatase deficiency in investigation 183

of steroid acids 174-176 of steroid conjugates 176--184 of steroid fatty acid esters 172-174 of steroid sulphates 179-184 of vitamin D metabolites 601 thermospray of triazoline adducts of vitamin D metabolites 585

use in studies of vitamin D metabolising enzymes 599

HRT see hormone replacement therapy human chorionic gonadotropin see testosterone and hCG

human pre-pro gene 567 hybrid mass spectrometer 105 hydrolysis

conjugates 428 enzymes 430

708

hydrolysis (contd) glucuronidase 430 fJ-glucuronidase 432 of estrogens 429, 430 steroid sulphatase 430

phosphate 430 saccharolactone inhibition 430

sulphatase 432 (IS)-I-hydroxy calciol 20 17-hydroxy corticosteroids 660 18-hydroxy-ll-deoxycorticosterone, assays for 256-257

IlfJ-hydroxyandrostenedione, assays for 247-248

17 fJ-hydroxy-5a-androstan-3-one 6 17 fJ-hydroxyandrost-4-en-3-one 6, 11 4-hydroxyandrostene-3, 17 -dione, HPLC-MS of 172

4-hydroxyandrostenedione, metabolites of in urine 122

3fJ-hydroxyandrost-5-en-17-one 6, 11 I1-hydroxyandrosterone 660, 690 25-hydroxy cholecalciferol 20 18-hydroxycorticosterone metabolites in urine 254 assays for 253-254 interaction with alcoholic solvents 253

18-hydroxycortisol, assays for 254-255 17 fJ-hydroxyestr-4-en-3-one 19 3-hydroxyestra-I,3,5(1O)-trien-17-one 5 3-hydroxyestra-l,3,5,7,9-pentaen-17-one 5 4-hydroxyestradiol 453, 456 2-hydroxyestradiol 453, 456 2-hydroxyestradiol plasma 450 2-hydroxyestradiol-17 fJ 449 15a-hydroxyestriol plasma 450 15a-hydroxyestriol serum 450 15a-hydroxyestriol-BSA 450 2-hydroxy estrogen 431 2-hydroxyestrogens 449 2-hydroxyestrone 14, 428, 449 2-hydroxyestrone plasma 450 2-hydroxyestrone-17 -( O-carboxymethyl)-[125IJiodohistamine 449

2-hydroxyestrone-17-( O-carboxymethyl)­oxime-BSA 449

ll-hydroxyetiocholanolone 660, 690 23-hydroxy-24-homo-la,25-dihydroxyvitamin D3 mass spectrum of per-trimethylsilyl

ether 605 structure of per-trimethylsilyl ether 605

I1fJ-hydroxylase deficiency 246 21-hydroxylase deficiency, 17-hydroxyprogesterone as marker for 246

17-hydroxylase deficiency 251, 257 hydroxylase enzymes, in steroid synthesis 230

INDEX

18-hydroxylated corticosteroids, measurement by GC-MS 163

18-hydroxylated steroids, problems with HPLC of 151

hydroxylated vitamin D metabolites, assignment of structure by NMR 609

17{i-hydroxy-17a-methyl androgens conjugation of 483 demethylation of 481--482 epimerisation of 480--481 metabolism of 475,478--483 see also methandrostenolone, methandienone, stanozolol

17 {i-hydroxy-19-nor-17a-pregn-4-en-20-yn-3-one 19

21-hydroxypregn-4-ene-3,20-dione 7, 11 3fJ-hydroxypregn-5-en-2-one 11, 109 17-hydroxypregnenolone sulphate, HPLC-MS of 182

17a-hydroxy progesterone CAP PT scheme 666, 669 EQA serum concentration 666, 669 IQC materials 660 Murex EQA scheme 689 UK NEQAS participation 654 UK NEQAS performance 671, 674

17-hydroxypregnenolone assays for 249 17-hydroxyprogesterone

assays for 243-245 concentration in plasma 243 immunoassay using extraction 122 interference by sulphate conjugates in

assay of 121-122 measurement in saliva 245 use of solvent extraction to prevent interference 122

hydroxysteroid dehydrogenase 3-a 530, 533, 539 7-a 531 12-a 531

hydroxysteroids 5, 6, 7, 8, 11, 13, 15, 17, 19, 22, 48, 79, 84

3a-hydroxysteroid dehydrogenase, use in detection of bile acids during HPLC 155, 157

17 fJ-hydroxysteroid dehydrogenase 444 3fJ-hydroxysteroid oxidases 108 25 hydroxy vitamin D 660, 688 25-hydroxyvitamin D

immunoassays for 588 measurement by GC-MS 596 NMR of 608

25-hydroxyvitamin D-la-hydroxylase 566 25-hydroxyvitamin D3

in human plasma 565 enzyme immunoassay for 574 enzyme labelled immunoassay for 587 HPLC-MS of 172

measurement by HPLC 579-581 structure 568

25-hydroxyvitamin D3-1-hydroxylase, in vitro studies using GC-MS 599

25-hydroxyvitamin D3-24-hydroxylase, in vitro studies using GC-MS 599

25-hydroxyvitamin D 3 - 26,23-lactone, structure 568

la-hydroxyvitamin D 3, structure of 569

1251 433 125I-labelled steroid 438 1251 NaI see 1251 sodium iodide 1251 sodium iodide 443 125I-histamine 448 125I-label 441 ID-GC-MS see gas

chromatography-mass spectrometry idiometric assay 434 immobilised antibodies in steroid extraction 129

immobilised antigen 439 immunisation 435, 436 immunoaffinity see extraction of anabolic

steroids immunoassay 430

accuracy 205,206,213 acridinium derivatives 197 affinity 189, 190 alphatype 210 anabolic steroids for 493-497 anti-allotype 210 antibody 186

characterisation 189 occupancy 209 titre 190

antigen binding site 209 antigenic response 187 automation 653 avidity 190, 191, 192, 204 background 199 betatype 210 biotin/streptavidin 205 bridge recognition 192, 193 calibration curve 205 captured analyte 209 carrier molecule 187 chelates 200 chemiluminescence 195, 212

enzyme assay 198

INDEX

competitive binding assay 185, 189, 198 correlation 207 cross-reactivIty 192, 202

antisera in 496-497 data 661 design of immunogens for 495-497 direct assay 201 directory 652

disassociation 199 displacing agents 202 enhanced 198, 201 enzymes 194 epitope 186, 187, 209, 210 excess reagent 205 external quality assurance 207 extraction 201 fertile period 203 FITC 213 fluorescence 198 fluorescence polarisation 213 fluoroimmunoassay 198, 213 for cortisol 238 et seq. free steroids 207 hapten 187, 188, 209

709

homogeneous immunoassay 212 hormone replacement therapy 202, 204 idiometric assay 210 idiotype 210 immunisation protocol 188 immunogen 187, 192, 193 immunogenic 187 immunoglobulins 186 immunometric 208 incubation timing 233 interference 206 isoluminol 195, 212 labels 192, 199 lanthanides 199, 203 matrix effect 201, 202 miscellaneous controls 206 monoclonal antibody production 188 non-competitive assay 208, 210 non-radioisotopic labels 194, 212 non-separation assay 210, 213 non-specific binding 204, 207, 208 of vitamin D metabolites 588-591 ovarian stimulation 204 photodetectors 195 polyclonal 187 precision 205, 206, 208, 210, 213 premenstrual syndrome 204 protein binding agents 202 quality control 205 quenching 193, 200 radioimmunoassay 185 radioisotopes 193 receptors 185, 189 recovery 206 reproducibility 206 saliva 203 sample preparation 201 saturation analysis 185, 189 scintillation 193, 211 scintillation proximity assay 210 screening assays 190, 210 second antibody assay 205

710

immunoassay (contd) sensitivity 190, 205, 208, 213 separation methods 204 separation of bound and free hormone 233

separation procedures 233 serum binding proteins 185, 202, 208 SHBG 202 simultaneous assay 203 solid phase 205, 208 specific activity 193 specific binding 202 specificity 189, 190, 191, 201, 202,

213 standardisation 201, 650 standards 206 steric hindrance 209 steroid analogues 208 steroid conjugates 195, 202 steroid derivatives 192 two-site assay 209 types 652, 653 urinary metabolites 203 urine 203 validation 205

immunoradiometric assay 453 imprecision 649, 657 see also precision inaccuracy 649, 657, 662 see also

accuracy; bias INADEQUATE NMR spectra 91 incubation time 437 indirect assay 448 infertile women 448 infertility 427 infrared spectra 38 injection onto GLC, methods available

139-140 injection onto HPLC columns

153-154 INSTAND 690 interassay variations 231 interfaces for LC-MS 166 et seq. interference 440

lipid 440 paper fibres 442 steroids 441 urate 432

Internal Quality Control 656-662 analyte concentration 658 data presentation 661 materials 658-659, 660 principles 657 procedures 659

internal standard use in mass spectrometric measurements

of vitamin D metabolites 575 use of deuterium labelled metabolites

575

INDEX

internal standards 115 et seq. use of in C19 steroid quantification

310-315 ion detection in mass spectrometry 103 ion-exchange columns, use in steroid

extraction and separation 134-135 ion-pair chromatography, in HPLC 153 ion-pairing agents 453 ion separation 99 ion trap mass spectrometry 94, 100, 105,

159-160 ionisation, in mass spectrometry 93 IQC see Internal Quality Control isofiavenoid phytoestrogens 456 isotachysterol isomers

formation 581, 595 use in HPLC assay for 25-hydroxyvitamin D3 581

isotope dilution in mass spectrometry 94 mass spectrometry see GC-MS see also gas chromatography-mass spectrometry

Karplus equation 75 Keller-Liliani reaction 624, 625 ketoconazole

effects on TIE ratios, of 514-515 use of, in differentiation of biological outliers to athletes using testosterone 515

ll-ketoandrosterone 660, 690 ll-ketoetiocholanolone 660, 690 17-ketogenic steroids 660 17-ketosteroids 660 keyhole limpet haemocyanin antigenic protein 448

Kieselguhr, in steroid extraction 125 et seq.

14C-labelled steroid 438 3H-Iabels 449 Laboratoire National de Sante 690 lactones 49, 56 Landelijke Werkgroep voor Bindingsanalysen 690

lanthanide ions and chelates use in fiuoroimmunoassays 346-349 use of in detection of anabolic steroids in HPLC 299

lanthanide shift reagents in NMR spectroscopy 67

latex particles 434 LC-MS 94, 166-184 see also HPLC-MS

levonorgestrel 692 LH20 use in steroid extraction and separation 134-135

Lichrosorb DIOL 454 LIDIA see ligand differentiation immunoassay

ligand binding assay 433

INDEX

ligand differentiation immunoassay, use in pseudohomogenous RIA 351

linearity 663, 675 linked scanning in mass spectrometry 104 Lipidex 1000 529 Lipidex columns 432 Lipidex-DEAP 530, 535 Lipidex use in steroid extraction and separation 134--135

lipoprotein disruption before efficient extraction

572-573 vitamin D incorporation into 573

liquid chromatography-mass spectrometry 94, 99

liquid secondary ion mass spectrometry (LSIMS) 97

lithocholic acid 11 LNS see Laboratoire National de Sante long-range spin-spin coupling in NMR spectroscopy 77, 92

luminescence, enhanced 439 lumistero12, HPLC of 565 lumisterol3

formation in skin 563 UV spectrum 564

luteal function 448 LWBA see Landelijke Werkgroep voor Bindingsanalysen

lyophilisation 658, 666

magnesium oxide, minicolumns in extraction 120

magnetic particles 439 magnetic sector instruments 100 magnetic sector mass spectrometers

99-100 mass fragmentography, of vitamin D metabolites 596 et seq.

mass spectrometric fragmentation 108 mass spectrometric resolution 102 mass spectrometry 93, 97-100

characteristic fragmentations by EI 108 combined GC-MS (SIM) 305-309 comparison of with RIA results 353 data handling 103 definition of common terms used in

94--95 derivatisation

of hydroxyl groups 106-107 of oxo groups 107-108

derivatisation techniques in 105-109 electron impact ionisation 94, 96 fragmentation 108

HPLC (thermospray) 316-317 ion detection and data handling

103-104 ion separation 99-100 ionisation 95

711

magnetic sector instruments 100--101 metastable peak monitoring in 309 negative ions CI of vitamin D3 in skin

594 of vitamin D metabolites and structural

analogues 594 et seq. principle ionisation and sample

introduction techniques 95 quadrupole ion trap 100 quadrupole mass filter 100 resolution of ions 94, 100, 102-103 sample introduction and ionisation 93 stable isotopes use in 309-310,311-315 static F AB-MS of vitamin D

conjugates 594 tandem (or MS-MS) 315-316 terminology 94 thermospray 595 time-of-flight (TOF) analysers 100--101 types of 305-318 use in measurement of DNA sulphate

249 useful ions for monitoring in 305-308 using chemical ionisation 94--95, 97

materials certified reference 680, 681 External Quality Assessment 665-666 Internal Quality Control 658, 659, 660

matrix 650, 658 matrix-assisted laser desorption/ionisation (MALDI), in mass spectrometry 99

MC903 see calcipotriol Medical Devices Directorate 655 Medipolar ODS 454 medroxyprogesterone acetate 692

determination by GC-MS 411--412 determination by RIA 410--411 structure 370

melengesterol acetate, determination by HPLC 157

menopause 427 mesteralone 441 metabolites 442 metastable ions, in mass spectrometry 94 methandienone

ions of metabolite 499 phase I metabolism 480--482 phase II metabolism 483 solid-phase extraction 486 structure 471

methandrostenolone see methandienone methanolysis comparison with enzymic

hydrolysis/solvolysis 491

712

methenolone confirmatory analysis of 504-505

method comparison 655 definitive 657, 666 evaluation 650-656 reference 650, 657, 662, 666, 678-685 selection 650-656 trouble-shooting 655 validation 655

methodology for vitamin D analysis 598-599

2-methoxy estrogen 431 2-methoxy[3HJestrone 449 2-methoxyestrone 449 methyl ether, derivative for mass

spectrometry 106 2-methyl ether 449 methyloxime, derivative for mass spectrometry 107

metyrapone 245 micellar chromatography, in HPLC 153 micro particle immunoassay 439 micro particulate silica

in steroid extraction 125 et seq. in vitamin D metabolite extraction 572

micro titre plate assay 439 mifepristone

anti glucocorticoid 412 antiprogestin 412 determination by HPLC 413 metabolism 412-413 pharmacokinetics 412 structure 370

milk, vitamin D in 562 mineralocorticoid, biosynthetic pathway 230

mobile phases in HPLC 152-153 statistical optimisation 152

molecular events in target cells of 1,25-dihydroxyvitamin D3 566-567

molecular ion 94 in mass spectrometry 96

monoclonal antibodies 391, 397, 435, 437 monoclonal antibody, use in immunoassay

of 1,25-dihydroxyvitamin D3 589 MO-TMS derivatives see gas chromatography-mass spectrometry

mouse osteopontin gene 567 MP A see medroxyprogesterone acetate MS 158-184 MS-MS 160-161 see also mass spectrometry, tandem

Murex Quality Assessment Programmes 689

NaBH4 see sodium borohydride

INDEX

nandrolone biosynthesis of 510, 517 ester formulations of 475 immunoassay of 494, 496-497 liquid-phase extraction of 488 metabolism of 476-477,484,494-495,

516-517 methanolysis of conjugates of 491 misuse of 468, 470 structure of 471

National Institute for Biological Standards & Control 663

natural binding proteins 433 network of steroid reference laboratories

692-693 neutral steroids, HPLC of 169-172 NH2 cartridges, for separation of vitamin

D metabolites 577 NHz columns, use in purification of estrogens 127

NIBSC see National Institute for Biological Standards & Control

NMR high field spectra of steroid hormones

610 of calcipotriol 608 of 25-hydroxyvitamin D 607 of vitamin D metabolites 606-610

NOESY NMR spectra 91 nomenclature 10 non-competitive assay see idiometric

assay non-extraction methods 441 non-invasive methods 445 non-invasive sampling 456 non-protein bound cortisol 452 non-protein bound estradiol

centrifugal analysis 453 flowing dialysis measurement 453 men 452 rats 452 women 452

non-protein bound estrogens 451 non-protein bound steroid 444 A-nor-5il(-androstane 19 4-nor-5il(-androstane 19 19-nor-5il(-pregnane 19 24-nor-5fJ-cholan-23-oic acid 19 nor-steroids 18 norethisterone 692

determination by GC-MS 413 determination by RIA 413-414 metabolism 415 structure 370

19-norethisterone 19 norgestrel

determination by RIA 415-416 determination in milk 416

structure 370 19-nortesterone 19 19-nortestosterone see nandrolone novel application 439 nuclear accessory factor (NAF) 566 nuclear magnetic resonance see NMR nuclear magnetic resonance spectra 56 nuclear Overhauser effect 82

o-catechol 449 OCT

antisera to 593 see also 22-oxacalcitriol

octadecylsilane-bonded silica 529, 530 oestradiol see estradiol off-resonance decoupling in 13C NMR spectra 85

16o:-0H estrone 431 250HD see 25 hydroxy vitamin D 170HP see 17o:-hydroxy progesterone on-line mass spectrometry 432 optical rotation 36 optical rotatory dispersion 36 oral contraceptives 451 ouabain 623, 624, 625 outliers 667 ovulation 439 22-oxa-l,25-dihydroxyvitamin D3 see 22-oxacalcitriol

22-oxacalcitriol (OCT), structure of 569 oxidation 443 oximes

derivatives for mass spectrometry 107 in mass spectrometry 107

18-oxocortisol, assays for 254--256 24-oxo-23,25-dihydroxydihydrotachysterol, structure 604

3-oxo-4-ene-steroids, HPLC-MS of 169-172

6-oxo-estradiol-17 fJ 428 6-oxoestradiol 431 16 oxo-estradiol 431 oxo-steroids 13, 54, 84 17 -oxosteroids

assays for GC-MS 280-291 estimation of by Zimmerman reaction

274 formation and conjugation of 271-272 separation of by GC profiling 301-303

paper chromatography 136-137,430,431 in assay of 1,25-dihydroxyvitamin D3

578 partition chromatography 442, 445, 446 Patella vulgata, in steroid conjugate hydrolysis 124

perfluoroacyl esters, derivatives for mass spectrometry 106

INDEX 713

peroxidase 439 phase I metabolism of anabolic steroids 475--483

phase II metabolism of anabolic steroids 483--485

'phase switching' 575 in vitamin D metabolite extraction 573

photodiode array detection, use in identification of steroids after GLC 155

phthalates interference in GC-MS of vitamin D metabolites 602

interference in mass spectrometry 587 phytoestrogens 441 placental insufficiency 445 placental sulphatase deficiency, HPLC-MS in investigation of 183

plasma concentrations of steroid hormones 231 of vitamin D metabolites by GC-MS

595 et seq. plasma estrogens 428 plasma steroids, measurement by GC-MS 164--165

polarisation immunoassay, for cortisol 240

polycystic ovary syndrome, androstenedione production in 247

ponasterone-A 632 postpartum blues 371,416 potato breeding, steroid analysis for 638-640

precision 438, 649, 659 see also imprecision

precision profile 659 prednisolone, HPLC of 145, 146 prednisone, HPLC of 145, 146 pregnancy 434

urine 447, 449 pregnane 2, 4 (20R)-5f:1-pregnane-3o:,20,21-triol 17 (20S)-5fJ-pregnane-3,20-diol 17 pregnanediol 17,660,690

determination by GC-MS 417 determination in milk 417 determination in serum 416--417 detennination in urine 372 glucuronide 692

5o:-pregnanedione see dihydroprogesterone

pregnanetriol 660, 690 metabolite of 17-hydroxyprogesterone

245 pregnanolone 399--400, 416 pregnant mare 450 pregnenolone 11, 14 5-pregnen-3fJ-ol-20-one-trimethylsilyl ether-methyloxime, EI mass spectrum of 109

714

Premarin 451 preservatives 658, 666 see also lyophilisation; sodium azide

previtamin D2, HPLC of 565 previtamin D3 562 PROBIOQUAL 690 Process Control see Internal Quality Control

Product Cycle 652 Proficiency Testing 662, 663, 691 see

also External Quality Assessment progesterone 7, 11, 86

antisera to 376-377 biosynthesis 291 CAP PT scheme 691 l1o:-carboxymethyl ether 398 determination of

by bioassay 372 by competitive protein binding

374-375 by double isotope derivative 374 byenzymeimmunoassay 385-389 by fluorimetry 373 by fluoroimmunoassay 390 by gas-liquid chromatography

373-374 by radioimmunoassay 375-385 by spectrophotometry 372-373

EQA serum concentration 666, 668 GC-MS for validating assay of 166 11o:-glucuronide 384, 397 l1o:-hemiphthalate 383-384 l1o:-hernisuccinate 376, 382-384, 397 12-hemisuccinate 382-383 IQC materials 660 metabolites, use of solid phase cartridges

for separation 133 Murex EQA scheme 689 solvent extraction 378-379 UK NEQAS participation 654 WHO EQAS 692

progestogens 369, 426 see also progesterone; medroxyprogesterone acetate; norethisterone; norgestrel

protein binding agents for disruption of 121

proton NMR spectroscopy 59 provitamin D3 see 7-dehydrocholesterol pseudohomogenous RIA for testosterone

350-351 PT see Proficiency Testing

QA see quality assurance quadrupole ion trap 100 quadrupole mass filter 100 quadrupole mass spectrometer 94, 99-100, 102

quality 647, 648

INDEX

quality assessment see External Quality Assessment

quality assurance 403-404, 438, 648 for 25-hydroxyvitamin D3 575, 587

quality control 403 see also Internal Quality Control

quality goals 648, 656, 662 quality manual 656 quality system 656

RAC see repeat analytical controls radioimmunoassay 324-332, 349-353

alternative methodologies for 332-334

commercially available kits for 324-332, 337

comparison of with GC-MS 353 of cyproterone acetate 407-408 of norethisterone 413-415 of norgestrel 415-416 of plasma progesterone 375-385 of salivary progesterone 396--398 pseudo homogeneous for testosterone

350-351 separation of bound/free steroid in

350-351 use in measurement of aldosterone 250-252

see also RIA radiolabelled steroids

as internal standards 117 use III Immunoassay 232, 233

Randox International Quality Assessment Scheme 689

rare earth chelates 439 rate dialysis 394 reagent gas 97 receptor auxiliary factor (RAF) 566 receptors for 1,25-(OH)2D3

in skin 567 widespread distribution 567

recovery 438, 663, 675, 678, 679, 683 5o:-reductase deficiency 242 reference interval 657 reference method see method refractive index, as method of detection during HPLC 154

repeat analytical controls 659 Repetends 659 see also repeat analytical controls

reproducibility 659 9-cis-retinoic acid 567 retinoid-X receptor (RXR) 566

involvement in tight VDR-VDRE binding and transcriptional activation 567

RIA 430, 433, 446, 456 see radioimmunoassay

INDEX

ring closure, in vitamin D metabolites during GC 594

ring residues in UV spectroscopy 31 R/S-nomenc1ature for chiral centres 20-23 RU486 see mifepristone

saliva analysis, as alternative to plasma 237 collection of 237 determination of progesterone in

396--398 equivalence to plasma free levels 396 17-hydroxyprogesterone in 245

saponification 120 scintillation counting 452 scintillation proximity assays 351, 433, 437 for cortisol 240 use in measurement of 1,25-dihydroxyvitamin D3 591

use of in eliminating separation of free/ bound steroid 351

screening analysis for anabolic steroids by GC-MS 498-503 by immunoassay 493-498 of natural hormones 509-518 of synthetic hormones 492-503

seco steroids 3, 20 (5Z, 7 E)-(lS,3R, 24R)-9,10-secocholesta-

5,7,10(19)-triene-1,3,24,25-tetrol 20 (5Z,7 E)-(3S)-9,1 0-secocholesta-5, 7,10(19)­

triene-3,25-diol 20 (5Z, 7 E)-(3S)-9, 1 0-secocholesta-5, 7,1 O( 19)­

triene-3-ol 20 (5Z, 7 E)-( 1 S,3R )-9,1 O-secocholesta-

5,7,10(19)-triene-1,3,25-triol 20 secocholestane 2 second antibody separation 433, 443, 448

in immunoassay 387 see also separation of free and bound

analyte SECSY NMR spectra 91 selected ion monitoring 94, 104 selected reaction monitoring 94, 104 Sep-Pak 572 Sep-Pak cartridges 432, 453 Sep-Pak SIL cartridges

for steroid purification 575-576 use in extraction of vitamin D metabolites 598

separation of free and bound analyte 443

dextran-coated charcoal 433 second antibody 433 solid phase 433, 437

of ions in mass spectrometry 99 separators for mass spectrometry 94 Sephacryl, use in affinity columns 574

Sephadex 431 Sephadex diethylaminoethyl 428 Sephadex G-25 429

715

Sephadex LH-20 430, 431, 441, 442, 443, 445,449,450,451,577

Sephadex, use in fractionation of neutral steroids 134

Sepharose-4B 445 sequence rules 21 serum see materials sex hormone binding globulin see SHBG SHBG 428, 430, 441, 451, 452 SHBG-bound testosterone, assays of 337-339

SHBG-effect in 'direct' assays for testosterone 336-337

silanisation of glassware 573 silica cartridges for removal of excess derivatising agents 577

silica-gel 442 silyl ethers in mass spectrometry 106 skin

effect of UV radiation 563 formation of lumisterol3 563 formation of tachysterol3 563 formation of vitamin D3 562 vitamin D3 in 594

SKZL see Stichting Kwaliteitsbewaking Klinische Chemische Ziekenhuislaboratoria

sodium azide 658, 665-666 sodium borohydride 445 soft ionisation 98

techniques in MS 159 fj-solamarine 637 solasodine 638 solid-phase antibody 442 solid-phase columns, use for separation of corticosteroids 13 3

solid-phase extraction 124 et seq., 572 details of available sorbents 126

solid phases Amberlite XAD2 use of 277-278 CIS by Sep-Pak cartridges use of 279 Extrelut use of 292 use in C 19 steroid extraction 277-279,

280-291, 324-332 solvent extraction 428, 443

of vitamin D and metabolites 571-572 selectivity of 121

solvent residues 440 solvents 440

available for steroid extraction 119 for IR spectroscopy 42 for NMR spectroscopy 66 for UV spectroscopy 29

solvolysis 429, 446 sorbents, for solid-phase extraction 126

716

specific rotation 36 specificity 435, 438, 441, 443 spin decoupling in NMR spectroscopy 81 spin-spin coupling in NMR spectroscopy 69,75

spironolactone 52 stable isotope labelled steroid, use of as internal standard in GC-MS 311-315

stable isotope labelling 108 stacked plot in 1 H NMR spectroscopy

88 standardisation 650 stanozolol

phase I metabolism of 478--480 phase II metabolism of 483 structure of 471 see also 17 fJ-hydroxy-171X-methyl androgens

stationary phases for GLC 137-138 steady state gel filtration 393-394 steric hindrance 433 steroid

acids, HPLC-MS of 174-176 analogues, as internal standards 116 binding proteins, interaction and extraction 114-115

conjugates, HPLC-MS of 176--184 hydrolysis of 122-124

derivatisation prior to GLC 140 fatty acid esters, HPLC-MS of

172-174 glucuronide profiles, in urine by negative ion LCIMS 98

glucuronides, enzyme hydrolysis 122-124

hydrazones, use in HPLC 156-157 sulphates

acid hydrolysis 122-124 HPLC-MS of 179-184 in plasma of newborn infants 244

steroidal alkaloids 637-640 Stichting Kwaliteitsbewaking Klinische Chemische Ziekenhuislaboratoria 690

stimulation and suppression tests 232 streptavidin 439 sulphatases 547 sulphate 15, 437, 439 17 fJ-sulphates

chemical synthesis of 482 epimerisation of 482 hydrolysis of 489--491 preferential formation of 483--484 quantification of intact conjugates of 492

urinary excretion of 476, 485 sulpho-conjugates see 17 fJ-sulphates sulphuric acid conjugation 449 Supe1co C18 454

INDEX

symmetric dialysis (SYD), saliva testosterone assay by 338-339

synergism 438 synthetic corticosteroids, assays for 243

tachysterol2, HPLC of 565 tachysterol3

formation in skin 563 structure 563 UV spectrum 564

tandem mass spectrometry 95, 104, 160-161

TIE ratio see hormone ratios testosterone 6, 11, 60, 86, 87, 437, 452

and hCG, effect on TIE test 515 and ketoconazole administration of

514-515 assays for conjugates 345 biosynthesis of 268 CAP PT scheme 691 CIAs for 346 cross-reactions of with 51X-DHT 340,

351 detection of administration of

in equine 518 in human 510-515

ELISAs for 340-341 EQA serum concentration 666,669 FIAs for 347-348 forms of in plasma, assay of 337-339 formulations of 475 hydrolysis of conjugates of 489--490,

511 hydroxy derivatives of, separation by HPLC 297-298

hydroxylation of 272 IQC materials 660 mass spectrum of

as MO-HFB 509 as oxime-bis-TBDMS 502

measurement of 337-339 by GC-MS 280-291,309

metabolism of 269-272, 476--477, 484--485

misuse of 470, 510 Murex EQA scheme 689 pseudohomogeneous RIA for 350-351 RIAs for 350-351 separation of, from epitestosterone and

llfJ-hydroxyandrosterone 511-512 specificity of tests for doping with

512-515 structural modifications of 474 total ion chromatogram for 512 UK NEQAS participation 654 urinary metabolites of, in equine 518 WHO EQAS 692 see also hormone ratios

tetrahydroaldosterone, measurement in urine 252

tetrahydrocortisol 690 tetrahydrocortisone 690 tetrahydrocortisone-3-glucuronide, HPLC-MS of 178

tetrahydro-ll-deoxycortisol 246 tetrahydro-18-hydroxy DOC 256 thermospray 95

HPLC-MS, use in analysis of steroid sulphates 248-249

mass spectrometry 95, 97, 99 MS 166--167

thin-layer chromatography 135-136 in separation of androgens 274--276,

280-291, 324--332, 336 in separation of C 19 steroid conjugates

319 of anabolic steroids 489 review of use 136 see also TLC

thyroglobulin-antigenic protein 447 time-of-flight analysers 101 time-resolved fluorescence 439 tissue extraction 121 TLC 430,431,442,443 TMS derivatives and anabolic steroids 498-500

total ion current 103 in mass spectrometry 103

Total Quality Management 648 TQM see Total Quality Management transfec1ed COS cell, use in assessment of biological activity 593

trenbolone structure of 471 triazoline adducts, use in measurement of

vitamin D metabolites 583-585 1,24,25-trihydroxyvitamin D3, structure 568

11{3,17,21-trihydroxypregn-4-ene-3,20-dione 7, 11, 86

trimethylsilyl ether, derivatives for mass spectrometry 106

trimethylsilyl ethers, formation from vitamin D metabolites prior to GC-MS 597

trimethylsilyl oximes, derivatives for mass spectrometry 107

tritium NMR spectroscopy 91 two-dimensional NMR spectroscopy

86 tyrosine 438 tyrosine methyl ester 443

UK NEQAS advisory panels 663 cortisol methods 651 data presentation 670

INDEX

GC-MS studies 678-685 linearity studies 675 oestradiol method types 653 of cortisol 238 participation 653, 654, 664 performance criteria 670

717

performance evaluation 667, 671-675, 676

recovery studies 677-678, 679 reports 670 samples 665 steering committee 663 surveys 664 target values 667, 671, 672, 673 urinary free cortisol 685, 687-688

ultrafiltration 394 ultraviolet

absorption spectra 26 detection of vitamin D metabolites after

HPLC 579 unconjugated estrogens 428 1X,{3-unsaturated ketones, detection after

HPLC 154-155 urinary steroids

analysis by LSIMS 98-99 assay in investigation of steroid abnormalities 236-237

in normal man by GLC 142 profile by GC-MS 162

urine estrogens 428 human pregnancy 431,432 profile analysis 242

uterus cytosol binding rabbit 430, 441, 442 rat 445

UV spectrum of 7-dehydrocholesterol 504 of lumisterol3 564 of tachysterol3 564 of vitamin D3 564

vaginal cornification 433 V AR see variability of the bias variability of the bias 670 see also UK

NEQAS, performance criteria vibrational spectra 38 vitamin D 8, 20, 33, 562 et seq.

assay of metabolites by saturation analysis 588-591

assays, use of internal standard 574

binding protein see DBP chemical analogues of 569 chemistry 562 et seq. esters 573 extraction 571-575 in food, extraction of 120

718

vitamin D (contd) in milk 562 measurement 581

by GC-MS 596 metabolites, assay by mass spectrometry 594 et seq.

metabolites, formation of N-O­methyloximes for GLC 595

metabolites, in plasma by GC-MS 596 et seq.

metabolites, measurement by HPLC with electrochemical detection 586

metabolites, separation on SIL cartridges 576, 577

metabolites, solid-phase extractions 572-573

metabolites, storage under nitrogen 573-574

metabolites, structural investigations by NMR 606-610

metabolites, structures 568-569 metabolites, use of affinity column for

extraction 574 metabolites, use of amber glass for

storage 574 metabolites, use of triazoline adducts in

measurement 583-585 nomenclature 562 receptor (VDR) 566

INDEX

reviews of analytical methodology for 571

solvents used for extraction 572 stages in analysis of 570 structure 562 et seq.

vitamin D-responsive element 567 vitamin D 2, in plants 562 vitamin D3

conjugates, use of static F AB 583 formation in skin 562 UV spectrum 564

vitamin D3-25-hydroxylase 565 vitamin D3-25-hydroxylase, in vitro studies

using GC-MS 599

Wakosil 5C18 454 walk-away automation see automated immunoanalysers

Wellcome Quality Assessment Scheme see Murex Quality Assessment Programmes

WHO see World Health Organisation withaferin-A 626, 627 withametelin 628 withanolides 626-631 World Health Organisation 663, 691-692

Zorbax SIL, use in HPLC separation of vitamin D2 isomers 565