Method for the Measurement of Prostaglandin F, in Biological Fluids by Gas Chromatography-Mass Spectrometry R. W.
Kelly
Medical Research Council, Reproductive Biology Unit, 2 Forrest Road, Edinburgh, EH7 2 0 W, Scotland
A method for the GC-MS analysis of prostaglandin F 2 a in biological fluids is described. The prostaglandin is extracted, and the extract is derivatized and injected directly into the gas chromatograph. The derivative chosen is the methyl ester 9,ll-butylboronate 15-trimethylsilyl ether: this derivative is selective and confers good gas chromatographic properties. The linearity of the mass spectrometer has been demonstrated over a 1OOO:l dynamic range. Concentrations of prostaglandin F 2 a down to 1 ng/ml can be measured with 20% relative standard deviation. The relative standard deviation for concentrations greater than 5 ng is better than 8 % . The accuracy of the technique has been proved.
The use of the mass spectrometer as a specific detector for the gas chromatograph provides one of the few physical methods which is sensitive enough to measure prostaglandins. The recent synthesis of deuterium-labeled prostaglandins ( 1 ) has made this type of analysis more practical and reliable. Despite the recent development of relatively specific radioimmunoassays for prostaglandins, physical techniques are still necessary to confirm measurements, especially in samples where prostaglandins have not heretofore been measured. Gas chromatography-mass spectrometry (GC-MS) techniques have been used previously for quantitation of prostaglandins in purified extracts ( 2 - 4 ) , but this approach is laborious and the final measurements do not take full advantage of the specificity of the GC-MS combination. This report describes a technique for GC-MS analysis of a suitable derivative of prostaglandin F z a which is specific and is sensitive enough to measure concentrations down to 1 nanogram per milliliter of plasma. In the method described, a crude extract is treated to make the derivative which is injected directly into the combined gas chromatograph-mass spectrometer. This technique is applicable to measurements in a wide range of biological fluids; it is suitable for semen, sheep plasma, uterine fluid, and for most human plasma samples; however, after a meal, fatty substances in the plasma sometimes interfere and the mass spectrometer has to be tuned to an ion of higher mass.
EXPERIMENTAL Extraction a n d Derivatization. Blood samples were collected on ice, centrifuged a t -4 "C within 15 min, and the plasma was stored a t -15 "C until required for analysis. Twenty microliters of a standard solution (3.8 ng/pl) of 3,3,4,4-tetradeutero prostaglandin Fzo,(kindly supplied by U. Axen of the Upjohn Co.) was thorU. Axen, K . Green, D . Horlin, and 6 .Samuelsson, Biochern. Biophys. Res. Cornrnun.. 45. 519 (1971). K . Green, E. Granstom. and B. Samuelsson, in Prostaglandins in Fertility Control," Vol. 2 , World Health Organization Conference, Stockholm. 1972. p 9 2 . L. S. Woife and C . Pace-Asciak, in "Prostaglandins in Fertility Control," Vol. 2. World Health Organization Conference, Stockholm, "
1972. p 201 L. Baczynskyj, D . J. Duchamp, J. F. Chem.. 45, 479 (1973)
Zieserl, J r . , and
U.
Axen. Ana/.
oughly mixed with a 5-ml ,sample of plasma. The plasma was acidified to p H 3 and then extracted with 20 ml of ether/ethyl acetate ( 4 : l ) . The aqueous layer was discarded and the ethereal layer washed to neutrality and then evaporated to dryness under vacuum a t 45 "C. The residue was transferred to a small tube and dissolved in 80 p1 of ethanol/ethyl acetate ( 1 : l ) . Two 25-11 aliquots were evaporated in 2 x 60 mm glass tubes and treated with diazomethane solution (25 11). The diazomethane was prepared from N-methyl-N-nitroso toluene-4-sulfonamide in ether and freshly distilled into methanol before use. The resulting solution was approximately 1% diazomethane in ether/methanol 2: 1. After 10 minutes, the diazomethane and solvent were evaporated and 30 p1 of a solution of butylboronic acid (Applied Science Laboratories) was added a t a concentration of 5 mg per ml in acetone/benzene 2 : l . The tubes were placed in a vacuum oven a t 60 "C a t a pressure 200 mm Hg below atmospheric. After 20 minutes under these conditions, the pressure was lowered to evaporate the solution completely and then 25 p1 of a 20% solution of bis(trimethylsily1)trifluoroacetamide (BSTFA) in hexane was added. After two hours, the BSTFA solution was evaporated under vacuum and 7 11 of a 5% BSTFA solution in hexane was added and the tubes were sealed in a flame. The total contents of the tube were injected into the GC-MS instrument. The derivatives were stable at room temperature for u p to 10 days and considerably longer a t -15 "C. In routine determinations, duplicate aliquots were analyzed, leaving a third aliquot for running as a different derivative if required. GC-MS Analysis. Gas chromatography was carried out on a Hewlett-Packard 402 gas chromatograph; 5-ft G shaped columns, packed with 1% Dexsil 300 on Gas Chrom Q, were used a t a temperature of 240 "C. Injections were made directly onto the column with no flash heater. The effluent from the GC column was passed through externally-heated glass lined stainless steel tubing to a Watson-Biemann separator and then into an M S 1 2 mass spectrometer (Associated Electrical Industries Ltd., Manchester). The temperatures of all lines between the GC column and the M S source were maintained at 250 "C; the separator was kept at 260 "C, and the source block at 240 "C. The mass spectrometer settings for routine use were as follows: ionizing voltage, 20 eV; accelerating voltage, 8 kV; ionizing beam current, 100 pA; electron multiplier voltage, 2.2 kV; resolution 600 (10%). The required masses were brought into focus by switching the accelerating voltage with an A.E.I. switching unit a t a fixed magnetic field. The dwell time on each channel was 0.5 second. The head amplifier of the spectrometer was replaced by a varactor bridge amplifier and the output from this was decoded to give separate signal channels for each ion monitored, as described elsewhere ( 5 ) .The signal from each ion was passed through a low pass filter with a first order cut-off from 0.2 Hz. The smoothed analog data from each ion were fed to two channels of a Rikadenki 6 channel pen recorder (type KA). The two channels were used a t different sensitivities to increase the dynamic range of the system. Quantitation was achieved by measuring the ratio of the peak height of the prostaglandin Fzo peak to that of the deuterated compound. Standard mixtures were interspersed with the samples and these were used to check the calibration from day to day. Once the ratio of the response of a known amount of PG F z n to that of an identical amount of deuterated equivalent was known, this figure could be used to calculate results directly from the ratios observed in extracts.
RESULTS Accuracy. The linearity of response of the GC-MS system was checked over a 1000-fold range by preparing standard mixtures which contained a constant amount of deu(5) R . W . Keliy, J. Chrornatogr.. 71, 337 ( 1 9 7 2 ) .
A N A L Y T I C A L C H E M I S T R Y , VOL. 45,
NO. 12,
OCTOBER 1973
2079
I . ""I
, 0
19
2n9 WT
200ng
2bn9
PROSTAGLANDIN F 2 a INJECTED
Figure 1. Linearity of response of the mass spectrometer detec-
0 0
tor Crossed lines denote the range within a series and superscripts give the number of samples. Triangles denote deviations from linearity when the contribution of the natural prostaglandin to the deuterated channel is ignored. Solid circles represent the mean response corrected for this contribution (8%) and corrected for a blank corresponding to a ratio of 0.01
2
4
lDDE0
6
8
Figure 2. Accuracy of measurement system in analyzing levels
prostaglandin F 2 a in pools of peripheral plasma from hysterectomized sheep which have a known amount of F?, added
of
Results express a means f std dev ( N = >6) 0 933x r = 0 989 for the line is y = 0 20
+
Table I. Measurements on Different Aliquots of Plasma Composition of sample. ml Plasma
Water
0
5 3 2 1.5
2
3 3.5
Prostagiandin measured in sample, ng
Calcd concn in plasma
82.0 49.4 35.0 26.6
16.4 16.5 17.5 17.7
10
P R O S T A G L l N D l N [t~g,/ml.]
The regression equation
terated standard (40 ng) and a variable amount (0.2 to 200 ng) of prostaglandin Fz,; the results are shown in Figure 1. The plotted values have had a background corresponding to a ratio of 0.01 subtracted from them to display linearity on a log-log scale. Above 20 ng Fz,, correction has to be made for an 8% contribution of the natural Fza to the deuterated channel. When this correction is made, linearity is restored for the full 1OOO:l dynamic range.
Table I I. Comparison of Radioimmunoassay with GC-MS Measurements Concn by GC-MS, ngiml
Sample
Sheep uterine vein plasma samples between day 1 5 and 17 of the cycle
Concn by R I A , ng/ml 8.9 5.8 1.8 9.2 3.5
10.4 7.4 2.5 7.7 3.6
uterine vein plasma, replicate determinations Uterine vein plasma from ovariectomized sheep Sheep uterine fluid Pool of
1.76
(mean,n = 6 )
1.56
(mean, n = 8 )
1.62 178
(mean,n = 6 )
1.42 223
(mean, n = 2 )
8
Table II I . Precision of Measurement in Replicate Samples Sample
Peripheral plasma from hysterectomized sheep with added prostaglandin
Uterine vein plasma Plasma from ovariectomized sheep Sheep uterine fluid 2080
Mean level of, No. of Sam- Precision, prostaglandin ples in Yo re1 F 2 a , ng/ml series std dev
9.2 4.8 2.9 1.2 1.76
6 7 6 6
20.0
1.62 178
6 6
5.5 1.3
7
9.1 4.6 12.6 10.0
The accuracy of the analytical procedure was checked in three ways. First, known amounts of Fzn were added to plasma pools from hysterectomized sheep and 5 m l aliquots of these pools were analyzed; the results of these analyses are shown in Figure 2. The regression equation of 0.933 x and the correlation coeffithis line is y = 0.20 cient is 0.989. Second, a sample of sheep uterine venous plasma (day 15 of the cycle) was successively diluted and the original and diluted samples were assayed. The results of these assays are shown in Table I and they confirm the accuracy of the technique for samples rich in prostaglandin Fza Third, the results of assays on plasma samples were compared with those obtained by radioimmunoassay (6). The comparison is shown in Table 11. The correlation
+
(6) B V Caldwell. S A Tillson, W A Brock and L Speroff Prostag/and!ns 1, 21 7 ( 1972)
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 12, OCTOBER 1973
i--------.-----L
l?
--. 8 RETENTION
RETENTION
TIME
(MINUTES)
Figure 3. An analysis of a typical low level sample of sheep uterine venous plasma ( 2 ng/ml prostaglandin F2a) Conditions are as in Figure 4; the arrows show the retention time where prostaglandin FZs is expected to elute and demonstrate the offset of the recorder pens
coefficient is 0.954 for values below 10 ng/ml, which is significant a t the 0.1% level. Precision. The precision of the method was checked on samples with known amounts of added prostaglandin and on pools of fluids known to contain prostaglandin FzU.The results of these determinations are shown in Table 111. The precision starts to fall rapidly below 1 nanogram, although with sheep plasma and uterine fluids, results can be obtained at the 0.2 ng/ml level with precisions on the order of 30-40% (relative standard deviation). The precision from extracts compares favorably with the precision of pure samples; a t the level of 5 ng of F z a injected, the relative standard deviation for pure standards is 5.3% ( n = 13). Recovery. The recovery was checked over the entire process of extraction by comparing the absolute peak heights from the deuterated material in standard mixtures with that of the recovered deuterated material in fluid extracts. These comparisons were made over a large number of samples to correct for variations such as loss on sample injection, change of sensitivity, etc. The mean recovery is 60%.
DISCUSSION Specificity. In any GC-MS measurement technique, the specificity of the gas chromatograph is compounded with that of the mass spectrometer. The criteria for interference by an impurity in this technique are that the compound must chromatograph as a peak which is within approximately 5 seconds of the maximum of the prostaglandin F z a peak ( i e . , it must have a retention time within approximately 1% of that of the prostaglandin) and must either give a relatively strong ion a t the m / e value moni-
4
0
TIME [ M I N U T E S )
Figure 4. A trace of an analysis of a sample (sheep uterine fluid) with a high level of prostaglandin F p U (178 n g / m l ) The tetra-deutero internal standard is monitored at m i e 439 in the bottom trace and the prostaglandin F*n is monitored at m j e 435 in the middle trace. The total ion current (TIC) monitor trace i s shown at the top; this trace is not required in the analysis but is added here to illustrate the specificity of the technique. Arrows show the point of injection on the three traces and give an indication of the offset of the recorder pens and the different starting points of the chromatograms
tored for the Fza channel or a very strong ion in the deuterated channel. Maximum use has been made of these elements of specificity and with the chosen derivative, the result is a sensitive and specific method which can be used for the measurement of trace components in biological extracts. Examples of the specificity of the tuned ion detector in this application are shown in Figures 3 and 4. Choice of Derivative. The prostaglandin Fzo, derivative chosen was the methyl ester 9,ll-butylboronate 15-trimethylsilyl ether, which allows good gas chromatographic performance and provides a reasonably intense peak in the mass spectrum. This derivative exhibits minimal adsorption and is more stable than the corresponding 9,11,15-tris(trimethylsilyl)ether. The use of a cyclic derivative such as a butylboronate adds specificity to a gas chromatographic analysis partly because immediate distinction is made between cis and trans diol compounds (7, 8). The mass spectrum of the butylboronate derivative (Figure 5) shows only one strong peak above m/e 320 ( a t mle 435). The relatively high mle value of this peak is an extra advantage because, in general, the higher the mass monitored, the lower the chance of ions from other compounds interfering in the measurements. Although the contribution of the 435 peak to the total ion current is only 11%, this is relatively high for a high molecular weight ion. In cases where the plasma is heavily contaminated with lipids, the derivative has to be changed; here the tris(trimethyl silyl) ether is used and the ion monitored for pros(7) C. J. W. Brooks and J. Watson, Chem. Commun , 1967,952. ( 8 ) C. Pace-Asciak and L. S. Wolfe. J. Chromatogr.. 56, 129 (1971)
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Figure 5. The mass spectrum above m / e 340 of prostaglandin F 2 a , methyl ester 9,ll-butylboronate 15-trimethylsilyl ether. The large peak at m / e 435 (A4 - 71) is due to the loss of the pentyl side chain (c16-c20)from the parent ion
taglandin Fza is m / e 513 ( M - 71). Monitoring a t this high value removes virtually all interfering peaks. When this derivative is used, the sensitivity of the method falls; but measurements can still be made a t the 1 ng/ml level with reasonable precision. Accuracy. The linearity of response in a GC-MS measurement system cannot be assumed, since complications such as beam broadening due to interionic repulsions will begin to affect the accuracy a t high sample levels. In this procedure, a linear response has been demonstrated with this mass spectrometer. It is important to establish a linear range that includes the likely concentrations of standard, if this standard is to be used for correction of losses during an analytical procedure. No deviation from linearity was detected when varying volumes of one plasma (a sheep uterine vein sample with 16 ng/ml of prostaglandin Fza) were assayed (Table I). This demonstrates that no interference from plasma is encountered during the extraction stage. The accuracy is further confirmed by comparison of the results from this method with results from a radioimmunoassay method (Table E). The correlation coefficient for values up to 10 ng/ml of 0.954 is compatible with the precision limits of both analyses. The precision data show that analyses can be performed down to 2 ng/ml with a precision of 6-10% (relative standard deviation). At the 1 ng/ml level, the precision is around 20%, although this is an upper limit and in some clean samples measurements can be made well below the 1 ng/ml level. When a sample of about one nanogram is injected into the chromatograph, the random low frequency noise (
