Biomedical .Mass Spectrometry 1976. !. I 1 7 to 121

Measurement of Prostaglandin F2a in Human Cerebrospinal Fluid by Single Ion Monitoring? H. THERESE CORYand P. T. LASCELLES Institute of Neurology, Queen Square, London W C l N 3BG. England

B. J. MILLARD$ The School of Pharmacy, Brunswick Square, London WC1 N 1AX, England

W. SNEDDEN and B. W. WILSON Department of Chemical Pathology, St. Bartholomew’s Hospital, London ECI, England (Received 9 December 1975) Abstract-An improved method is described for measuring low levels of prostaglandin F1a in small volumes of cerebrospinal fluid. Single ion monitoring of the ion at m/e 413, derived from the methyl ester tri-TMS ether derivatives of prostaglandin F1a and the internal standard, o-trinor- 16-cyclohexyl prostaglandin F2a, is employed. Injection of 7 pg of prostaglandin F2a derivative gave a standard deviation of +9”i,. The majority of patients had cerebrospinal fluid levels of up to 2 ng ml-’ of prostaglandin F2a, whereas in certain neurological conditions with progressive disease, elevated levels were encountered, some of which were higher than iongrnl-’.

Introduction

measured PGF2a in CSF, using a known excess of 3,3,4,4-d4-PGF2cr as both carrier and internal standard, utilizing selected ion monitoring of mle 423 and 427. By this method, it was possible to obtain a standard deviation of 2302 for eight injections of 1OOpg of PGFZa. It is more sensitive and precise to monitor a single ion rather than two or more.’ For single ion monitoring, the best internal standard is a homologue of the compound being measured, yielding an ion of the same mie value. The two compounds should be adequately resolved by the g.c. column. Unfortunately, small amounts of compound injected into g.c. columns may adsorb onto binding sites present in the packing material, or decompose on the surfaces of the g.c.m.s. interface. The extent of the loss varies considerably with the type of column, the nature of the compound and the design of the instrument. For this reason, the use of a deuteriated analogue in large excess, solely to act as a carrier, may be desirable. We have applied the concept of using added deuteriated carrier plus homologue to the measurement of PGFZcr, and a preliminary account of the method has already been published.’

THEGROWING interest and speculation regarding the physiological r61e of various prostaglandins in animal tissues-the central nervous system in particular-has led to the demand for more sensitive and specific techniques for their measurement. Although bioassay and radioimmunoassay have been used successfully to measure prostaglandins,‘,2 these do not possess adequate specificity. Ambiguities can arise in distinguishing between the ten or so naturally occurring prostaglandins and their metabolites in animal tissues. Since these compounds have closely related structures, yet possess differing, sometimes contrasting roles in the same tissues, it is of critical importance to be certain that the compound of interest is being measured. While it is possible to detect prostaglandins in picogram amounts by these other methods. they are less sensitive than the gas chromatography mass spectrometry (g.c.m.s.) technique, which can measure femtogram quantities of prostaglandin F2a ( P G F ~ x ) .Since ~ , ~ the levels of PGFZa in human cerebrospinal fluid (CSF) are thought to be less than 1 ng ml-‘ in normal subject^,'.^ and only small amounts of CSF are normally available, g.c.m.s. appears to be the most suitable method for measurement. The measurement of prostaglandins by g.c.m.s., using selected ion monitoring, was first reported by Samuelsson et al.’ In this method, a deuterium labelled analogue was used to act as a carrier for the natural prostaglandin through the g.c. system, and as an internal standard for quantification. Wolfe and Mamer6

t Abbreviation: P G F h

Experimental CSF SAMPLES

Spinal and ventricular fluids were assayed from 37 patients admitted to the wards of a specialized neurological hospital. For ethical reasons, it was clearly not possible to obtain CSF from normal subjects: however, CSF was taken and assayed in a double-blind form. The samples available for the analysis of prostaglandin were usually between 0.5 ml

= prostaglandin F2a.

1Present address: Institute of Neurology, Queen Square, London WClN 3BG, England.

0 Heyden & Son Ltd. 1976

117

118

t

H. T. CORY, P. T. LASCELLES, B. J. MILLARD. W. SNEDDEN AND B. W. WILSON

and 2 ml in volume: the remainder of the samples were used for the performance of routine diagnostic tests. The CSFs were immediately cooled to 4 "C after collection, and, within 5 h, centrifuged at lo00 g for 5 min, to sediment any cells present, which might lyse upon freezing and release additional prostaglandin or prostaglandin metabolizing enzymes. The supernatants were transferred to clean vessels. which were sealed and stored at -20°C until used. The time between collection and thawing of the samples for extraction of the prostaglandins ranged from 2 weeks to 2 months.

deactivated by injection with 10 x 2Opl portions of Sylon BTZ (Supelco Inc.) and preconditioned at 290 "C for 48 h before use. Before each run, 2 ng of standard PGF2a derivative was injected to saturate the binding sites., Mass spectrometric running conditions Ionizing voltage, 90 eV (nominal) ; ionizing current, 3 mA: resolving power (10% Valley), 900: single ion monitoring of m/e 423 for both PGF2a and homologue derivatives. Under these conditions, 1.0 pg of PGF2a derivative injected onto the g.c. column gave a signalto-noise ratio of greater than 50: 1.

EXTRACTION

The solvents used in the extraction procedure were of analytical grade. Methanol and ethyl acetate were redistilled before use, the latter over anhydrous potassium carbonate to remove traces of acid. Glassware was treated with 'Dri-Film' (Pierce Chemical Co.) to minimize losses by adsorption. A measured amount of CSF (1 ml or less) was mixed with 9 m l of ethanol, and 100ng each of 3,3,4,4-d,PGF2a and internal standard (w-trinor- 16-cyclohexyl PGF2a) in 0.1 ml of methanol were added. When sufficient CSF was available, extractions were performed in duplicate. The rest of the procedure was essentially as described elsewhere.2 The dried residues of the prostaglandin extracts were stored in sealed containers at - 20 "C until analysed. On the day of analysis, the dried extracts were converted to their methyl ester tri-TMS ether derivatives as already d e ~ c r i b e d . ~ STANDARD CALIBRATION CURVE

.

The apparatus was calibrated by preparing standard mixtures, in which 100 ng of tetradeuteriated carrier and internal standard (homologue) were added to differing amounts of PGF2a. The range was chosen to cover the concentrations at which PGF2a might be expected to occur in the CSF samples. The mixtures were derivatized and analysed in the same way. In order to establish that the internal standard and the PGF2cr co-extracted to the same extent, standard mixtures were also taken through the same extraction procedure as the CSF samples above.

Results and discussion STANDARDS AND CALIBRATION

Figure 1 shows a selected ion recording of m/e 423 for two consecutive injections of 1.Opg of PGF2a. The peaks due to the derivatized prostaglandin and internal standard are well separated. Figure 2 shows a plot of the ion current ratios for the PGF2a and internal standard derivatives at m/e 423, against the amount of PGF2cr injected in the presence of a constant amount of internal standard. Each mixture was prepared in duplicate, and each sample injected at least four times. The curve is linear over the range measured, but it intercepts the y-axis at a point above the origin. Although the response factors of the PGF2a and internal standard derivatives will not be identical, it is assumed that they remain constant for the duration of a measurement. RECOVERY EXPERIMENTS

In order to establish that the internal standard and the prostaglandin were both recovered to the same extent when taken through the extraction procedure, standard mixtures were made up in duplicate. in which 100 ng each of deuteriated carrier and internal standard were added to 1.0,5.0 and 10.0 ng of PGF2a in 1 ml of methanol. These solutions were taken through exactly PGF2o

PGF2u

GAS CHROMATOGRAPHY MASS SPECTROMETRY

Analyses were carried out using a Varian MAT 31 1A mass spectrometer, coupled to a Varian Aerograph 1440 gas chromatograph by means of a two-stage BiemannWatson separator. Details of the g.c. and m.s. running conditions have been presented but some important parameters are as follows. L

Gas chromatographic running conditions The glass column (2 m by 3 mm bore) was packed with1 % SE 30 Ultraphase on Supelcoport ; column temperature, 260 "C : injection port, 280 "C : carrier gas, helium ; flow rate, 45 ml min- '. The column was

4

3

2

1

0

4

3

2

1

0

Time (rnin)

FIG. 1. Two consecutive 1 pI injections of lOpg P G F k and 2OOpg of internal standard as methyl ester tri-TMS ether derivatives. Single ion monitoring of mje 423.

PROSTAGLANDIN F2z IN CEREBROSPINAL FLUID

119

is thought to be due to allylic proton transfer under the influence of the 5 :6 double bond. On the basis of this finding therefore, we decided to abandon the use of the carrier for the analysis of the present series of CSF samples. These were extracted with the addition of 100ng of internal standard only. RECOVERY AND PRECISION FOR SAMPLES EXTRACTED WITH NO ADDED CARRIER

01

Y i

0

I

10

20

30

40

-

PGF20 injected (pg)

FIG. 2. Response ratios at mie 423 for PGF2a to internal standard vs amount of PGF2a injected with constant amount (500pg) of internal standard. The methyl ester tri-TMS ether derivatives are used. (a) Synthetic mixtures in methanol; (b) Synthetic mixtures extracted by the same procedure as the CSF samples.

the same extraction procedure as the CSF samples and derivatized as described. The ratios of the peak heights of PGF2a to internal standard are shown in Fig. 2(b). It can be seen that when these figures are plotted on the same axes as the standard calibration curve. they fall on a straight line which has a slightly lower slope than the first curve. It was therefore concluded that though the yields of PGF2a and internal standard may be reduced in the course of the extraction procedure, the ratios of the peak heights remain the same as those measured when the mixtures were not extracted. In calculating the actual concentrations of PGF2a in our CSF samples, (b)was used as the reference curve, and any values falling above the upper limit of the curve were listed as > 10 ng ml-

'.

MODIFICATION OF PROCEDURE

During the course of the work, it was found that 'blank' specimens, containing deuteriated carrier and internal standard but no PGF2a, gave peaks of increasing height at the retention time appropriate to PGF2a. Over a period of four weeks, the blank increased to an amount equivalent to 10-20% of the amount of deuteriated carrier originally added, this being roughly ten times the quantities of PGF2a expected in the samples. An examination of the mass spectrum of a methanolic stock solution of the deuteriated carrier (normally stored in methanolic solution at 4°C) showed that in addition to the fragment ion at mje 427 characteristic of the tetradeuteriated compound, ions of comparable intensity were present at mle 426, 425, 424 and 423. indicating that proton exchange had taken place. No such exchange was found in a more concentrated specimen of the tetradeuteriated compound which had been stored at - 20 "C. The exchange

It was now necessary to appraise the precision of our modified method, using single ion detection in the absence of carrier: Accordingly, mixtures of PGF2a with a known excess of internal standard were derivatized and analysed. For injections of 2, 5 and 1Opg of PGF2a, the standard deviations were 9 % (8), 10 % (5) and 12% (5) respectively, the figures in parentheses denoting the number of injections. These standard deviations are of the same order of magnitude as previously obtained using added ~ a r r i e r . ~ Now that it was established that the omission of the carrier did not decrease the reproducibility of the responses obtained in injecting very small samples into our g.c.m.s. system, it still remained a possibility that the multistage extraction of small amounts of PGF2a in CSF in the absence of carrier would result in greater prostaglandin loss, due to adsorption on glassware and other factors. We had at our disposal one 10 ml sample of post-operative CSF. Accordingly, mixtures of 1 ml of CSF and 100ng of internal standard, plus known amounts of PGF2a were prepared in duplicate, extracted, derivatized, and analyzed as before. Each sample was injected three times. The mean values of the response ratios are listed in Table 1. T-LE 1 Response ratios for PGF2z vs internal standard after derivatization Concentration of PGF2a in the mixture (ng per 100 ng internal standard)

Amount of PGF2z injected

(Pg)

0 1

0

2

10

5

Response ratios: PGFZajinternal standard 0.093 0.130 0.159

These ratios are very close to the values which would be predicted from examination of the standard calibration curve (Fig. 2). Therefore, it appears that the omission of the carrier does not impair the recovery of small added amounts of PGF2x through the extraction procedure. BLANK VALUE

Samples containing no deuteriated PGF2cr and no PGF2a but only internal standard (5 pg) gave peak height ratios equal to the value at which the standard calibration curves (a) and (b) in Fig. 2 intersected the y-axis (0.10 20% (5 injections)). Therefore, it appears that the small amount (less than 0.05 %) of interfering substance is present in the internal standard, since

H. T. CORY, P. T. LASCELLES, B. J. MILLARD, W. SNEDDEN AND B. W. WILSON

120

injection of blank silanizing reagent showed no response at the retention time appropriate to PGF2a. Therefore we decided. for the present series of CSF samples, to express very low levels of CSF prostaglandin as being less than 0.5 ng ml-’.

SPECIFICITY O F RESPONSE AT mle 423 FOR PGF2z

Measurements were performed to exclude the possibility of a contribution to the response at rn/e 423 by compounds other than the fragment ion from the PGF2a derivative (see Ref. 4). At a resolving power of

TABLE2. Levels of PGF2z in cerebrospinal fluid

Patients Name/sex/age

CSF findings Total proteinjglobulin testiblood cell count

1. E.A.-A.(m)

Normal

65 Y 2. M.A. ( f ) 40 Y

Normal

3. C.A. (f) 27 Y 4. T.C.(m)

56 y Operarioe ventricular

CSF 5. M.G.-C. (f) 47 Y

Total protein normal Globulin abnormal Ceil count normal Ventricular CSF not analysed : for spinal C S F : Total protein raised Cell count raised Total protein raised Globulin abnormal Cell count raised Normal

6. M.G. ( f ) 26 y 7. E.G.(m) 8Y (a) Ventricular Total protein normal Globulin normal CSF Cell count raised (b) Ventricular CSF, 2 days after (a) (c) Spinal C S F , Total protein raised 3 days Globulin normal Cell count raised after (a) 8. N.H.(m) Normal 60 Y 9. P.H.(m) Normal 65 Y Normal 10. D.H.(m) 31 Y Total protein raised 11. H.K. (m) Globulin abnormal 52 Y Cell count raised Normal 12. G.K.(m) 43 Y Total protein raised 13. D.M.(m) Globulin abnormal 12 Y 14. M.O’S. ( f ) Normal 46 Y Total protein raised 15. V.R.(m) Globulin abnormal 54 Y Normal 16. C.R.(f) 28 Y Normal 17. L.R. ( f ) 22 Y Normal 18. J.R. ( f ) MY 19. P.R. (m) Normal 48 Y

Final diagnoses Secondary New growth Multiple sclerosis Inactive phase Multiple sclerosis Cerebral cyst

Fungal meningitis Syringomyelia

Meningitis

PGF2a levels ng ml0.8 1.o