Direct derivatization of sulphate esters for analysis by gas chromatography mass spectrometry

Direct Derivatization of Sulphate Esters for Analysis by Gas Chromatography Mass Spectrometry?$

Stephen Murray and Thomas A. Baillies Department of Clinical Pharmacology, Royal Postgraduate Medical School, Ducane Road, London W12 OHS, UK

Gas chromatography mass spectrometry has been used to study the reaction between a range of aromatic, alicyclic and aliphatic sulphate esters and three perfluoroacylating reagents, viz. trifluoroacetic, pentafluoropropionic and heptafluorobutyric anhydride. Aromatic sulphates were found to react readily, when treated with a 1 : 1 mixture of a perfluoroacid anhydride and dry ethyl acetate, to form the perfluoroester derivative of the parent phenol in quantitative yield. Non-aromatic sulphates, on the other hand, gave a variety of products. A mechanism is proposed for the one-step derivatization of aromatic sulphates and the potential of the procedure for the analysis of sulphate conjugates by mass spectrometry is discussed.

INTRODUCTION

The formation of sulphate conjugates has long been considered to be a terminal stage in the metabolism of many endogenous compounds and xenobiotics to afford excretory products with little or no biological activity.’ Although this may often be the case, there are numerous exceptions;2 thus, morphine 6-sulphate has recently been shown to be a more potent analgesic agent than free m ~ r p h i n e , ~ while steroid sulphates are known to play an important role in both the biosynthesis and metabolism of steroid hormones, when they may undergo a variety of transformations without cleavage of the sulphate g r o ~ p . ~ . ~ Furthermore, in contrast to the generally accepted role of sulpho-conjugation as a ‘detoxication’ mechanism in drug metabolism, esterification with sulphate appears to be a key step in the conversion of several N-hydroxylated arylamines to highly reactive electrophilic species which bind covalently to cellular macromolecules leading, in turn, to mutagenesis and carcinogenesis.6

Increasing interest in sulphate esters has led to a requirement for improved analytical methods which may be employed for the characterization and quantitative measurement of trace amounts of these polar compounds in extracts of biological origin. Gas chromatography mass spectrometry has played an important

t A preliminary account of this work was presented at the American Society for Mass Spectrometry, 25th Annual Conference on Mass Spectrometry and Allied Topics, Washington, DC (1977).

i Abbreviations and systematic nomenclature: TFAA = trifluoroacetic anhydride; PFPA = pentafluoropropionic anhydride; HFBA = heptafluorobutyric anhydride; BSA = N.0-bis-(tri- methylsily1)acetamide; TSIM = trimethylsilylimidazole; terbutaline = 1 -(3,5-dihydroxyphenyl)-2-(f-butylamino)ethanol; androsterone = 3cr-hydroxy-5a-androstan- 17-one; epiandrosterone = 3P-hydroxy- 5a-androstan-17-one; dehydroepiandrosterone (DHEA) = 3p- hydroxy-5-androsten- 17-one; pregnanediol = 5P-pregnane-3a,20a - diol; tetrahydrocortisol = 3a, l lp,17a,21 -tetrahydroxy-5P-pregnan- 20-one; MHPG = 3-methoxy-4-hydroxyphenylethylene glycol.

0 Present address: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, Califor- nia 94143, USA.

role in this connection, although an indirect approach is mandatory, whereby the sulphate ester is first hy- drolysed to the parent alcohol, which is then converted to a suitable volatile derivative. While a more direct approach would be preferable, the thermal instability and non-volatile character of intact sulphate conjugates has precluded their analysis by conventional mass spectrometric methods, although spectra of representative examples have been obtained using laser ionization7 and field d e s ~ r p t i o n ~ ’ ~ techniques. Despite the need for methods of converting, in high yield, sub-microgram amounts of sulphate esters into volatile derivatives for GCMS analysis, only limited progress has been made in this

Hydrolysis of sulphate esters is normally carried out by the action of either hot acid or sulphatase enzymes. These procedures, however, suffer from a number of disadvantages, e.g. the use of hot acid is necessarily restricted to sulphates of compounds which are them- selves stable at low pH, while commercially available sulphatase preparations are often crude, and therefore non-specific, and may contain impurities which interfere in the final analytical step. Although solvolysis (mild acid) conditions have been described which minimize the extent of artefact formation,12 the development of alternative procedures for the cleavage of sulphate esters under mild conditions is clearly desirable.

Following a number of observations that certain sulphates undergo reaction with silylating reagents to give TMS ethers of the corresponding phenols directly,13-16 Touchstone and Dobbins17 reported on a similar reaction between steroid sulphates and heptafluorobutyric anhydride (HFBA). In view of the potential usefulness of this ‘one-step’ approach for the direct conversion of sulphate conjugates into volatile derivatives suitable for mass spectrometric analysis, we have investigated the scope of the reaction by examining the behaviour of a range of aromatic, alicyclic and aliphatic sulphates towards some derivatizing reagents commonly used in GCMS work, viz. trifluoroacetic anhydride (TFAA), pentafluoropropionic anhydride (PFPA) and HFBA. The results of this study indicate

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82 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, -1979 @ Heyden & Son Ltd, 1979

DERIVATIZATION OF SULPHATE ESTERS FOR GAS CHROMATOGRAPHY MASS SPECTROMETRY

that the reaction products obtained depend markedly on the nature of the sulphate ester employed, the aromatic sulphates being the most reactive of the three groups investigated.

EXPERIMENTAL

Chemicals

Derivatizing reagents were purchased from the following suppliers and were used without purification: acetic anhydride (BDH, Poole, UK), TFAA (Sigma, Kingston- upon-Thames, UK), PFPA (Pierce & Warriner, Ches- ter, UK) and HFBA (Koch-Light, Colnbrook, UK). A

Potassium 4-nitrophenol 1-sulphate (1)

CH,O OH OH

Potassium MHPG 4-sulphate (4)

sample of acetic anhydride, free of acetic acid, was obtained by fractional distillation of the commercial material.

Reference samples of sulphate esters, together with the corresponding parent alcohols, were mainly of commercial origin. Terbutaline and its (phenolic) sulphate conjugate were gifted by Dr E. Hansson and 3a-hydroxy-Sa-[3P,ll,l l-2H3]pregnan-20-one sulphate by Professor J. Sjovall. Professor D. N. Kirk donated the following steroids from the MRC Steroid Reference Collection: androsterone, androsterone 3- sulphate, epiandrosterone, epiandrosterone 3-sulphate, pregnanedio17 pregnanediol 3-sulphate, 3a-hydroxy- 5a-pregnan-20-one; tetrahydrocortisol and tetrahydrocortisol 21-sulphate. The salt forms in which the sulphate esters were obtained are indicated below.

~ . -

Dipotassium 4-nitrocatechol 1 -sulphate (2) Hydrogen terbutaline 3-sulphate (3)

[K]' -0 ,SO

Potassium estrone 3-sulphate (5) Sodium estriol 3-sulphate (6)

Sodium androsterone 3-sulphate (7) Potassium epiandrosterone 3-sulphate (8) Sodium DHEA 3-sulphate (9)

Sodium pregnanediol 3-sulphate (10)

CH ,(CH,), ,CH,OSO,- "a]'

Potassium 3a-hydroxy-Scu-[3f3.11 ,I l-2H3]pregnan-20-one sulphate (11)

Sodium dodecanol 1-sulphate (12)

@ Heyden & Son Ltd, 1979

Sodium tetrahydrocortisol 21 -sulphate (13)

BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979 83

S. MURRAY AND T. A. BAILLIE

Solvents (BDH ‘Analar’ grade) were dried and redistilled before use.

Gas chromatography

This was carried out using a Varian 1400 Series gas chromatograph, equipped with a flame ionization detector. Spiral glass columns (6 ft x 2 mm i.d.) were used, packed with either 3% OV-1 or 3% OV-17 on Gas Chrom Q (80/100 mesh; Phase Separations, Queensferry, UK). The flash heater zone and detector oven were maintained at 240°C and nitrogen (30 ml min-’) was used as carrier gas. Column tempera- tures used for the analysis of reaction products are given in Tables 1-3.

Gas chromatography mass spectrometry

A Finnigan Model 3200 GCMS instrument was used, equipped with a glass column (5 f t X 2 mm i.d.) packed with either 3% OV-1 or 3% OV-17 on Gas Chrom Q (100/120 mesh). The injector and separator tempera- tures were maintained at 260 and 250 “C respectively, and helium (30 ml min-’) was employed as carrier gas.

The mass spectrometer was operated in the EI mode, with an electron energy of 25 eV, emission current of 400 pA and electron multiplier voltage of 1.6 kV. Data acquisition and reduction was performed by a Finnigan Model 6100 interactive data system.

Derivatization procedures

The reaction between sulphate esters and perfluoroacylating reagents was investigated using the following general procedure. An amount of the sulphate equivalent to 20 bg of the parent alcohol was placed in a 1 ml ‘Reacti-Vial’ (Pierce & Warriner, Chester, UK) and was treated with ethyl acetate (100 pl) and the appropriate perfluoroacid anhydride (100 pl). The vial was firmly capped and the reaction allowed to proceed, either overnight at room temperature (aromatic sulphates) or at 70°C for 30min (non-aromatic sulphates). The reaction mixture was then evaporated under a stream of dry nitrogen and the residue taken up in ethyl acetate (50 pl) for analysis by GC and GCMS. The effect of pyridine on the reaction of aromatic sulphate esters towards TFAA was investigated using a similar procedure, in which the substrate was treated

Table 1. Gas chromatographic and mass spectrometric properties of products of the reaction between aromatic sulphate esters and perfluoroacid anhydrides

Compound

Nitrophenol sulphate

Nitrocatechol sulphate

MHPG sulphate

Terbutaline sulphate

Estriol sulphate

Estrone sulphate

Acid anhydride

TFAA

PFPA

HFBA

TFAA

PFPA

HFBA

TFAA

PFPA

HFBA

TFAA

TFAA

PFPA

HFBA

TFAA

PFPA

HFBA

Column temp.

(“C)

120

120

120

120

120

120

125

125

125

140

250

250

250

250

250

250

Retention time (min)

2.60

2.05

2.50

2.60

1.95

2.25

4.90

3.20

4.45

3.90

2.10

1.35

1.40

3.90

3.10

3.20

GC data (OV-171

Identity of reaction product

Nitrophenol TFA

Nitrophenol PFP

Nitrophenol HFB

Nitrocatechol bis-TFA Niirocatechol bis-PFP Nitrocatechol bis-HFB MHPG tris-TFA

MHPG tris-PFP

MHPG tris-HFB

57 Terbutaline (100) tetra-TFA

Estriol tris-TFA

Estriol tris-PFP

Estriol tris-HFB

Estrone TFA

Estrone PFP

Estrone HFB

a [MI’’ region out with mass range of Finnigan 3200 GCMS system.

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Table 2. Gas chromatographic and mass spectrometric properties of products of the reaction between alicyclic sulphate esters and perfluoroacid anhydrides

GC data (OV-17)

Acid Compound anhydride

Androsterone sulphate TFAA PFPA HFBA

Epiandrosterone TFAA sulphate PFPA

HFBA DHEA sulphate TFAA

PFPA

HFBA

Pregnanediol sulphate TFAA

PFPA

H FBA

[’H,]Pregnanolone TFAA sulphate PFPA

HFBA

” OV-I as stationary phase.

Column temp.

(“C)

220”

240

215”

215a

21

230

230

230

230

21 5

215

220

Retention time (min)

2.90’

3.00

4.65”

4.80”

5.40”

3.00

3.25

2.75

3.00

4.55

5.00

8.45

mfr values and relative abundances of characteristic ions

Fragment ions Identity of

reaction oroduct

218 190 161 147 122 106 93 Androsten-l7-one (97) (63) (100) (44) (72) (70) (75)

190 (53) 255 (13) 255 (15) 255 (17) 284 15) 284 15) 284 (4) 394 13) 147 (34) 161

(100)

161 147 122 97 93 (100) (33) (70) (50) (41) 213 199 121 107 105 (13) (23) (100) (43) (29) 213 199 121 107 105 (13) (23) (100) (40) (28) 213 199 121 107 105 (14) (23) (100) (43) (31) 255 215 121 108 93 (10) (20) (59) (100) (63) 255 161 121 107 93 (12) (93) (81) (100) (86) 215 121 108 93 81 (16) (53) (100) (49) (52) 215 161 121 107 93 (38) (100) (80) (83) (72) 121 108 93 81 (50) (100) (45) (47) 121 107 93 81 (79) (77) (62) (58)

Androsten-17-one

97 DHEATFA (25) 97 DHEAPFP

(25) 97 DHEAHFB

128) 81

(68) Pregnen-20-01 TFA 81 (91)

Pregnen-20-01 PFP 81

(74)

Pregnen-20-01 HFB

288 248 205 163 109 84 43 [*H3]Pregnen-20-one (11) (94) (78) (64) (66) (94) (100)

with 200 p1 of a mixture of ethyl acetate+pyridine+ TFAA (87.5 : 7.5 : 5 by vol).

The reaction between estrone sulphate and acetic anhydride was carried out as follows. An amount of estrone sulphate equivalent to 20 pgof free estrone was treated with acetic anhydride (50 p1) and ethyl acetate (150 pl) and the mixture, in a tightly-capped Reacti- Vial, allowed to stand overnight at room temperature.

Water (1 ml) was then added and, after thorough mixing, the product was extracted with ether (2 x 1 ml). The combined ether extracts were washed with 1 N HCl (2 x 1 ml), saturated NaHC03 solution (1 ml), dried (Na2S04) and evaporated under nitrogen. The residue was redissolved in ethyl acetate (50 1.1) for analysis by GC and GCMS. In studies on the effect of pyridine on the reaction with acetic anhydride, the substrate was

Table 3. Gas chromatographic and mass spectrometric properties of products of the reaction between aliphatic sulphate esters and perfluoroacid anhydrides

GC data (OV-171

Column Retention mlz values and relative abundances of

anhydride (“C) (min) [MI+’ Fragment ions Acid temp. time characteristic ions

Compound

Dodecanol sulphate TFAA 120 3.35 282 (0)

PFPA 120 3.05 332 (0)

Identity of reaction product

213 168 140 125 111 97 83 69 57 Dodecanol (2) (4) (8) (11) (32) (63) (87) (98) (100) TFA

213 168 140 125 111 97 83 69 57 Dodecanol (1) (5) (8) (12) (35) (63) (89) (94) (100) PFP

213 168 140 125 111 97 83 69 57 Dodecanol ( < I ) (5) 15) (6) (27) (53) (77) (82) (100) HFB

HFBA 120 3.40

Tetra hydrocortisol TFAA No volatile products formed sulphate PFPA

HFBA

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S. MURRAY AND T. A. BAILLIE

treated with a mixture of acetic anhydride + ethyl acetate+pyridine (50 pl: 50 ~ 1 : 100 pl) and the reaction and subsequent work-up procedure were carried out as described above.

In all cases, the extent to which a given sulphate was converted into the appropriate ester of its parent alcohol was assessed by GC analysis of the reaction product; the height of the peak obtained by injection of a fixed aliquot of the reaction product was compared with that obtained by injection of the same aliquot of a second solution, prepared by taking an authentic sample (20 Fg) of the parent alcohol through an identical derivatization procedure. Yields are quoted to the nearest 10%.

In addition, the availability of a deuterium labelled analogue of 3a -hydroxy-5a -pregnan-20-one sulphate permitted a direct comparison to be made of the reaction of the sulphate ester and the parent alcohol, present as a mixture, with a given reagent. In these studies, in which the substrate consisted of equimolar amounts of pregnanolone (unlabelled) and pregnanolone sulphate (deu- terated), analysis of the reaction mixture by GCMS indicated the fate, and rate of reaction, of the two molecular species.

RESULTS AND DISCUSSION

In the present study, we have compared the behaviour of a number of sulphate esters and their respective parent alcohols towards three homologous perfluoroacylating reagents widely used in the analysis of biological samples by GC and GCMS techniques.

All of the parent alcohols, with the exception of terbutaline and tetrahydrocortisol, reacted smoothly, at room temperature, with a 1 : 1 mixture of ethyl acetate and each of the perfluoroacid anhydrides to give a single derivative, whose identity as the expected fluoroester was confirmed by GCMS analysis. Terbutaline and tetrahydrocortisol gave multiple derivatives on reaction with all derivatizing reagents, although when the reaction mixture containing terbutaline and TFAA was heated for 1 h at 65 "C, the tetra-TFA derivative of terbutaline was obtained as sole product.

In contrast to the behaviour of the free alcohols, reaction of the sulphate esters with acylating reagents proceeded in a manner which was strongly dependent on the class of sulphate employed as substrate. Thus, aromatic sulphates reacted readily, at room temperature, with all three anhydrides to afford the perfluoroacyl derivatives of the corresponding parent alcohols in quantitative yield. Of this group, only terbutaline sulphate gave rise to multiple derivatives, paralle- ling the behaviour of unconjugated terbutaline. DHEA sulphate, however, was the sole example of an alicyclic conjugate which could be converted, in high yield, into a perfluoroester derivative of the parent alcohol (Fig. 1); the presence of a A5 double bond is known to enhance the reactivity of sulphate esters of 3P-hydroxy steroid^,'^.'^ although no direct evidence was obtained in the present investigation for homoallylic participation in the displacement of the sulphate group from the C-3 position." The major products formed from all other alicyclic sulphates studied were found to be unsaturated compounds, derived from the elimination of the ele-

7 ~ 7 -7 -7 1-7-7 7

6 4 2 0 6 4 2 0

Time (min)

7-77

6 4 2 0

'- 0 T ~- r 7 -7

6 4 2 0

Figure 1. Gas chromatograms recorded during the analysis (OV- 1) of the products of reaction between DHEA and TFAA (a), PFPA (b) and HFBA (c), and between DHEA sulphate and TFAA (d), PFPA (e) and HFBA (f). The peaks with retention times of 4.65,4.80 and 5.40 min correspond to the TFA, PFP and HFB derivatives, respectively, of DHEA, while the unresolved doublet at 2.9- 3.2 min, present in all chromatograms, corresponds to two isomeric androstadien-17-ones formed by elimination of the substi- tutuent at C-3.

ments of H2S04 from the conjugate. Injection of methanolic solutions of alicyclic sulphates directly into the GCMS system produced the same pattern of olefinic products, suggesting that they originated in the flash heater zone by thermal decomposition of the conjugates." Although it has been suggested" that these decomposition products may prove useful for the direct analysis of alicyclic sulphates by GCMS, the major disadvantage of such an approach lies in the multiplicity of isomeric olefins which are often produced.

Of the aliphatic sulphates examined, tetrahydrocortisol sulphate gave no volatile derivatives, either on reaction with any of the three anhydrides or on direct analysis by GC, thermal elimination of sulphuric acid being blocked in this case by the absence of a proton on the carbon atom adjacent to the site of conjugation. Dodecanol sulphate, on the other hand, did react, albeit somewhat slowly, with each of the anhydrides to give a perfluoroester derivative; interestingly, if reaction was incomplete (e.g. after overnight reaction at room temperature), or if a sample of dodecanol sulphate was injected directly into the GCMS system, the parent alcohol, dodecanol, was identified. The identities of reaction products obtained by treatment of aromatic, alicyclic and aliphatic sulphate esters with perfluoroacid anhydrides are listed in Tables 1, 2 and 3 respectively,



Table 4. Effect of time on the reaction (ambient temperature) of aromatic sulphate esters with peAuoroacid anhydrides. Results are expressed as percent theoretical yield of the fluoroacyl derivative of the parent alcohol, rounded off to the nearest 10%

Substrate Reaction

Acid time Nitrophenol Nitrocatechol MHPG Estriol Estrone anhydride Ih) sulphate sulphate sulphate sulphate sulphate

TFAA 4 100 80 60 100 100 8 100 100 100 100 100

PFPA 4 100 70 80 100 100 8 100 100 100 100 100

HFBA 4 100 100 80 100 100 8 100 100 100 100 100

which also summarize the relevant GCMS data. Semi- quantitative studies on the conversion of sulphate esters into fluoroacyl derivatives of the corresponding parent alcohols gave the results shown in Tables 4 and 5. Of the aromatic sulphates examined, only nitrocatechol sulphate and MHPG sulphate failed to react completely in 4 h at room temperature, although quantitative conversion was observed after 8 h. The apparently lower degree of reactivity of these two sulphate esters was attributed to their somewhat poorer solubility in the derivatizing reagents; in the case of MHPG sulphate, the use of a large excess of TFAA has been found to result in complete derivatization after a period of only 2 h at ambient temperature.21 For the non-aromatic sulphates which did afford fluoroacyl derivatives, viz. DHEA sulphate and dodecanol sulphate, solubility of the conjugate in the derivatizing reagent again appeared to be rate-limiting in the reaction.

In a series of experiments designed to establish the mechanism by which aromatic sulphates undergo ‘direct’ derivatization on treatment with acid anhydrides, estrone sulphate was employed as substrate. Acetic anhydride, rather than one of the perfluoroacid anhydrides, was selected for use as the derivatizing

reagent on the basis of its relatively low reactivity. The results of this study are given in Table 6 , which compares the behaviour of estrone sulphate and free estrone towards acetic anhydride, either alone or in the presence of acid (acetic acid) or base (pyridine). The well-known catalytic effect of pyridine on the acetylation of free estrone is evident from the results, whereas the presence of pyridine was found to strongly inhibit the reaction of estrone sulphate with acetic anhydride. Similarly, no reaction of the sulphate conjugate could be detected when freshly redistilled acetic anhydride was employed as reagent, although addition to this purified anhydride of a small amount (3% by volume) of acetic acid resulted in the quantitative conversion of estrone sulphate to estrone acetate. Clearly, therefore, the ‘direct’ derivatization of aromatic sulphates is an acid-catalysed phenomenon, which may be suppressed, either by addition to the reaction mixture of a base or by removal from the reagent of trace amounts of acid by distillation. Furthermore, comparison of the reaction products obtained by treating free estrone and estrone sulphate with unpurified acetic anhydride (or redistilled acetic anhydride to which a trace amount of acetic acid had been added) indicates that free estrone itself is unlikely to be an intermediate in the conversion of estrone sulphate to the corresponding acetate. Only when large amounts of acetic acid (30% by volume) were present did free estrone and estrone sulphate yield the same reaction products. The catalytic role of methanesul- phonic acid on the conversion of estrone sulphate to estrone acetate has been noted previously.22

Two distinct mechanisms for the acid-catalysed reaction of estrone sulphate with acetic anhydride, consis- tent with the above findings, are proposed in Scheme 1. At low acid concentrations, nucleophilic attack by the sulphate ester on a protonated acetic anhydride results in the expulsion of SOs and affords a complex which subsequently decomposes with elimination of acetic acid to give estrone acetate directly. Increasing the acid content of the medium, however, leads to protonation of estrone sulphate, which may first eliminate SO3 to give

Table 5. Reaction of non-aromatic sulphate esters with perfluoroacid anhydrides. Results are expressed as percent theoretical yield of the fluoroacyl derivative of the parent alcohol, rounded off to the nearest 10%

Substrate

Acid Derivatization Androsterone Epiandrosterone DHEA Pregnanediol [2H31Pregnanolone Dodecanol Tetrahydrocortisol anhydride Conditionsa sulphate sulphate sulphate sulphate sulphate sulphate sulphate

TFAA A B C D

PFPA A B C D

HFBA A B C D

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

70 100 100 60 70 I00 100 70 80 100 100 80

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

10 0 0 60 70 10 10 70 70 10 10 80

0 0 0 0 0 0 0 0 0 0 0 0

a Derivatization conditions were as follows: (A) 100 111 anhydride+ 100 PI anhydrous ethyl acetate, reacted overnight at ambient temperature; (B) 100 pl anhydride alone, reactedfor 30 rnin at 70°C; (C) 100 p,I anhydride+ 100 111 anhydrous benzene, reactedfor 30 min at 70°C”; (D) 100 pI anhydride+ 100 p,I anhydrousethyl acetate, reacted for 30 min at 70°C. In all cases, an amount of the sulphate ester equivalent to 20 kg of the parent alcohol was employed as substrate.

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S . MURRAY AND T. A. BAILLIE

Table 6. The effect of pyridine and acetic acid on the reaction of estrone and estrone sulphate with acetic anhydride. Reactions were carried out as described under Experimental

Reaction productis)

Estrone sulphate as substrate Estrone as substrate Reagent

No reaction Acetic anhydride”+ pyridine (1 : 1, V/V) Acetic anhydridea Estrone+estrone acetate (-1 : 1) Estrone acetate only

No reaction Redistilled acetic anhydride Redistilled acetic anhydride+ 3% acetic acid Estrone acetate only Redistilled acetic anhydride+ 30% acetic acid Estrone+ estrone acetate (-2 : 1)

a Commercial sample of acetic anhydride, used without purification.

Estrone acetate only

Estrone+estrone acetate (-1 : 1) Estrone+estrone acetate (-1 : 1) Estrone + estrone acetate (-2 : 1)

0 -q 11

0-AI I

0-S-O- Ar so3

+ 0-Ar CHS-C=O r+ I A\

A CH,-C=OH + CH3-C-&-H -+ +

H+

I I HOCOCH3 OCOCH3 /OCOCH3

0 0 I1 -2 1 1 y+ so3 II 0

B -0-S-0-Ar -+ 0-S-0-Ar + + I1 I ArOH O H

ArOH + (CH3C0)20 + ArOCOCH3 + CH3COOH

Scheme 1. Proposed mechanisms for the acid-catalysed reaction between aromatic sulphate esters and acetic anhydride. At low acid concentrations, mechanism A will predominate, whereas at high acid concentrations, mechanism B will be the major pathway.

free estrone; the unconjugated steroid formed then reacts slowly with acetic anhydride, resulting in the presence of both estrone and its acetate derivative in the final reaction mixture. The ‘direct’ derivatization of aromatic sulphate esters would thus involve the former mechanism (commercially available acid anhydrides invariably contain traces of the corresponding acid), while conventional acid-catalysed hydrolysis of sulphates is considered to operate via the latter pathway.18 Although these mechanisms are proposed on the basis of studies using acetic anhydride as reagent, the reaction of aromatic sulphate esters towards TFAA was also found to be inhibited by the addition to the medium of pyridine .or by the use of freshly purified reagent; these findings indicate a common mechanism for fluorinated and non-fluorinated acid anhydrides.

From the results of the present study, it would appear that the reaction of sulphate conjugates with perfluoroacid anhydrides, leading to the formation of perfluoroester derivatives of the parent alcohol in a single step, is a general reaction only in the case of aromatic sulphates. There may, however, be certain exceptions, such as the 3-sulphates of steroids with a 30-hydroxy-A5 structure. It is of interest in this connection that the steroid sulphates investigated by Touchstone and Dobbins17 were, with one exception (androsterone sulphate), either estrogens or 3 P - h ~ -

droxy-A5-androstenes, conjugated through the 3-hydroxy group. In our hands, androsterone sulphate failed to give a heptafluorobutyrate derivative, both under our own derivatization conditions and those of Touchstone and Dobbins; instead, an unsaturated product was obtained (Table 2).

Although we have confined our investigation to the reaction of sulphate esters with acid anhydrides, results obtained by other workers would suggest that certain silylating reagents, e.g. BSAI4 and TSIM,I6 behave in a similar fashion towards aryl sulphate conjugates to give TMS ether derivatives of the corresponding phenols. In view of the mild conditions under which such deriva- tizations may be performed, this approach would seem to hold particular promise for use with sulphates which cannot be analysed satisfactorily by conventional hydrolysis techniques. One such example is MHPG sulphate, for which we have developed a stable isotope dilution GCMS assay based on the conversion of the conjugate into the MHPG tris-trifluoroacetate derivative in a single step.” A key feature of this assay is the initial chromatographic separation on Sephadex LH-20 of MHPG sulphate from the free glycol, since treatment of the latter species with TFAA also yields MHPG tris-trifluoroacetate. In contrast, the glucuronide conjugate of MHPG is stable towards the derivatizing reagent.

The ‘direct’ conversion of aromatic sulphate esters into volatile derivatives suitable for GCMS analysis is a procedure which may be expected to find widespread application in the biomedical field, particularly in view of recent developments in methodology, e.g. ion- exchangez3 and ion-pairZ4 chromatography systems, for the isolation of these polar conjugates from biological sources. However, it should be noted that derivatization of crude extracts of biological fluids, which may contain both a metabolite of interest and its sulphate conjugate, may well lead to conversion of both species to a common derivative-quantitative results from GC-MS analyses carried out in this manner should therefore be treated with caution.

Acknowledgements

The authors would like to thank Professor J. Sjovall (Karolinska Institute, Stockholm, Sweden) and Professor D. N. Kirk (Westfield College, London, UK) for generous gifts of steroidsulphates, and Dr E. Hansson (AB Draco, Lund, Sweden) for a supply of terbutaline sulphate.



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@ Heyden & Son Ltd, 1979

@ Heyden & Son Ltd, 1979 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979 89

Direct derivatization of sulphate esters for analysis by gas chromatography mass spectrometry

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