The separation, identification and estimation of prostaglandins in nanogram quantities by combined gas chromatography-mass spectrometry

Vol. 9, Part I, pp. 989-988, 1970 . Life Sciences Printed in Great Britain

Pergamon Press

THE SEPARATION, IDENTIFICATION AND ESTIMATION OF PR03TAGLANDIN3 IN NANOGRAM QUANTITIES BY COMBINED GAS CHROMATOGRAPHY-MASS SPECTROMETRY C.J . Tho~mpsoa, M . Los and E .A . Norton Departaent of Pharmacology, University oß Edinburgh, 1 George Square, Edinburgh . Bß8 9JZ

(Received 26 June 1970; in final form 6 July 1970)

Prostaglandins are released ßrom aany tissues and organs either spontaneously or in response to nerve stimulation, hormones or drugs (1, 2) .

The quantity oß prostaglandin released and

available ßor analysis is frequently less than 1 microgram .

In

view oß these ainute amounts ideatißication has usually depended upon a combination oß chromatographic and biological evidence . Such evidence is inconclusive . There is thereßore a need ßor a method oß estimation which combines high sensitivity with more conclusive ideatißication . Bergstrôm, Samuelsson and their coworkers (3, 4) have made eztensive use oß mass spectrometry ßor the identification oß prostaglandins and their metabolites is animal tissues but the amounts analysed were greater than 1 microgram .

Recently,

sensitive gas chromatographic methods oß estimation have been described (5, 6), but with these methods also, ideatißication is inconclusive . lle now report a combined gas chromatographic-mass spectrometric method ßor separation, identification and estimation oß prostaglandins in amounts down to 10 naaograms .

This method

provides good evidence oß identification and has been applied successßully to prostaglandins in eztracts oß biological material . Derivatives oP the prostaglandins were prepared at room

989

984

QUANTIFICATION OF PROBTAGLANDINS

Yol . 9, No,17

temperature on a microecale using 0 .5 ml stoppered tubes .

All

samples were methylated by reaction ßor 20 min . with a ßreshly prepared solution oß diazomethane in diethyl ether-methanol (9 :1). Trimethylsilyl ethers were formed by the addition of 25 ~.1 bis silyltrißluoroacetamide (BSTFA) to the methyl ester .

10 ~.l

samples were injected on to the column after 3 hr without removal oß the BSTFA .

Trißluoroacetates were prepared by react-

ing the methyl eaters for 2 hr with 200 ~1 trifluoroacetic anhydride which was then removed in a vacuum desiccator . residue was dissolved in 25

The

ail hexane .

Analyses were performed on an LKB gas chromatograph - mass spectrometer, 10 ~1 samples oß the appropriate derivative being injected .

The column (1 .5 m z 1 .5 mm i .d .) was packed with 3',6

OV 1 on Supasorb Aw, 100-120 mesh pre-treated with dimethyl chlorosilane in carbon tetrachloride . ßlowed at 20 ml/min .

The carrier gas, helium,

All mass spectra were recorded at an

electron voltage oß 27 .5 . The trimethylsilyl ethers or the trißluoroacetates oß methyl esters oß proetaglandins F1~ and F AQ could be separated by our gas chromatographic system (Table 1) . Conclusive identißication was obtained by taking a mass spectrum oß the material is each peak .

The most abundant m /e

values characteristic ßor each prostaglandin derivative are shown in Table 1 .

By scanning at the appropriate retention time and measuring the heights oß prominent m/e peaks, it has been possible to estimate quantities down to 10 ng (Table 2), an amount which is subthreshold on the gas chromatographic record .

Even lower levës

oß prostaglandins are detectable from the mass spectrum but identißication becomes progressively less certain as the number of

Vol . 9,

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QUANTIFICATION OF PR05TAGLANDIN3

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Vol. 9, No.17

characteristic

QUANTIFICATION OF PROBTAGLAI~INS

987

n /e peaks which can be distinguished from noise

level aad background, diminishes . Although results have been presented in detail ßor prostaglandias Fia and Fsa only, these methods have been applied

successßully to nanogrsn quantities oß other prostaglandias .

The

trimethylsilyl ethers oß prostaglandias Ai, A~, Bi and Bs and their 19 hydroxy derivatives can all be separated by the same gas chromatographic syste= .

The trißluoroacetates oß prostaglandias

A (but not B) and prostaglandias E (which are converted to the corresponding A) can also be chromatographid. The eztractioa oß prostaglandias ßroa biological material involves solvent partition ßollowed by silicic acid column chromatography .

The latter separates prostaglandias into three

groups :- (1) prostaglandias A and H,

(2) prostaglandias E (3)

prostaglandias F and 19 hydroay prostaglandias A and B .

This

procedure does not however remove other acidic substances which invariably accompany the prostaglandin .

Analysis oß the

partially purißied samples by gas chromatography reveals many substances in addition to prostaglandias aad their metabolites . By measurement o! the characteristic peaks in its mass spectrum taken at the appropriate retention tine it is possible to identißy and estimate a known prostaglandin even in the presence oß these contaminants . Açkaowledgements we are grateßul to C .N . Goods ßor ezpert technical assistance and to R .L . Jones who isolated 18 hydroay prostaglandias Ai and A~ ßrom human seminal plasma and prepared the prostaglandin Bs ßrom prostaglandin E~ .

we are indebted to the Upjohn Company ßor

samples oß prostaglandias Ei, Es , Fia , Ai , Az and Bi, and to

988

QUANTIFICATION OF PR05TAGLANDINS

Vol . 9, No.17

Proßessor B . Samuelsson ßor 19 hydroxy prostaglandins B1 and B a . This research was supported by a grant ßrom.the Wellcome Trust . Reßerences 1.

3 . BERGSTROM, L.A . CARL30N and J .R . WEEKS, Pharmac . Rev. _20,

1 (1968) .

2.

E .W . HORTON, Phys ol . Rev . _49,

3.

S . BERGSTROM, R. RYHAGTs, B . SAMUELSSON aad J . SJOVALL,

122 (1969) .

J . biol . Chem . 238, 3555 (1963) . i 4.

M . HAMBERG, Europ . J . Biochem. _6, 135 (1968) .

5.

G .H . JOUVENAZ, D .H . NUGTEREN, R K . HERRTHUI3 and D.A . VAN DORF, Biochim . biophys . Acta _202, 231 (1970) .

6.

P .W . ALBRO and L. FISHBEIN

. Chromat . 44, 443 (1969) .