BIOMEDICAL CHROMATOGRAPHY, VOL. 6,24-29 (19%)
Quantitative Determination of Valproic Acid and 14 Metabolites in Serum and Urine by Gas ChromatographylMass Spectrometry E. Fisher, W. Wittfoht and H. Nau* Institute for Toxicology and Embryopharmacology, Free University Berlin, D-1000 Berlin 33, Germany
A method for the determination of the antiepileptic drug valproic acid and 14 of its metabolites in serum and urine by gas chromatography/mass spectrometry with selected ion monitoring of the trimethylsilylated derivatives has been developed. Sample preparation, including hydrolysis of VPA-conjugates and removal of urea in urine is carried out at pH 5.0 and is rapid and simple. The samples are extracted with ethyl acetate and the concentrated extracts are trimethylsilylated. Analysis with adequate separation of metabolites is achieved with a DB 1701 fused silica (Megabore) capillary column. The method exhibits high recovery and reproducibility and is sufficiently sensitive and selective for analysis of small sample volumes. Application of the method for screening patient serum and urine samples for unusual metabolite patterns, with possible predictive value for early detection of liver injury, is presented.
INTRODUCTION
Valproic acid (VPA) is a widely used antiepileptic agent with proven clinical efficacy for a variety of seizure types (Chapman et al., 1982). VPA is metabolized by a number of enzymes present in microsomes, mitochondria, peroxisomes and cytoplasma (Nau and Loscher, 1982, 1984). Some metabolites, most notably the unsaturated metabolites, including 2-en-VPA and 2,3 '-dien-VPA (see Scheme 1 for abbreviations), possess anticonvulsant activity in several animal models and may therefore contribute to the therapeutic effect of VPA (Liischer and Nau, 1985; Loscher et al., 1988); such compounds may also prove to be useful antiepileptic agents by themselves (Nau, 1986; Nau and Hendrickx, 1987). A potential for hepatic failure in patients maintained * Author to whom correspondence should be addressed.
on VPA, however, especially among a recently identified high-risk group, has made hepatotoxicity associated with VPA administration a major concern (Dreifuss et al., 1987; 1989). The mechanism of VPA-induced toxicity is still unknown, but a series of clinical and experimental findings have focused attention on the extensive hepatic metabolism of VPA (Eadie et al., 1988). Several lines of investigation indicate that idiosyncratic hepatotoxicity associated with VPA administration may occur secondarily to the formation of toxic metabolites in susceptible individuals (Grannemann et al., 1984; Kesterson et a l . , 1984; Baillie, 1988; Schafer and Luhrs, 1984). Monitoring the pattern of VPA metabolism during the course of therapy may therefore help to identify those patients in whom altered metabolism of VPA presents the risk of adverse effects. Due to the large number of metabolites of VPA and their structural sifnilarity (Scheme 1), a very specific assay is required for their simultaneous determination,
on
J
3-OH
a- alucuronkle (urine)
\
f
COOH
-
valproic acid(=)
\
3-keto COOH
2.4 -dm(E)
4-en -
4 - keto -
PGA -
Scheme 1. Structures and abbreviations of VPA and metabolites measured.
0269-3879/92/010024-06 $05.00 01992 by John Wiley & Sons, Ltd.
Received 8 January 1991 Accepted 1 March 1991
GC‘/MS OF VALPROIC ACID AND ITS METABOLITES
5-0q
9.41
2s
I
mle 199
mle 185 14-e“1
I/
13-
1
4-keto
miel95
3-keto-&en
mie 215
\
044
128
212
256
TIME(MIN)
340
044
424
128
212
256
340
424
TIME(MIN)
Figure 1. Selected ion chrornatograrns for G U M S analysis of untreated patient serum spiked with VPA and metabolites.
particularly because of the large dynamic range of concentrations present: VPA plasma levels are sometimes in excess of 100 pg/mL, whereas the concentrations of some metabolites can be below 100 ng/mL. Gas chromatography/mass spectrometry (GUMS) is therefore the chosen method because not only a host of VPA metabolites must be assayed, but these compounds must also be differentiated from endogenous compounds with related structures. Since our original GC/MS method was published in 1981 (Nau et al., 1981), a number of modified procedures have been developed (Rettenmeier et al., 1989; Tatsuhara et af., 1987; Abbott et al., 1986, Kassahun et d., 1989, 1990). All procedures appear to use our ethyl acetate extraction, although we adjust the pH of plasma and urine samples to 5.0 in order to avoid formation of multiple products of some hydroxy and keto metabolites; consequently, problems arise with these metaboTable 1 . Groups of ions selected to monitor characteristic fragments of trimethylsilylated VPA and various metabolites Scan
miz
Metabolites
1-300
197 199 201 215
4,4’-dien,2,4-dien,2,3’-dien 4-en,3-en; Z- and f-2-en VPA IS”
301-420
191 199 215
3-OH 4-OH 4-keto
183 3-keto 185 S-OH, PGA 195 3-keto-4’-en IS = internal standard (2-ethyl-2-methylhexanoic acid).
421-650 a
lites when the samples are acidified to lower pH values, as pointed out by others (Rettenmeier at al., 1989). The concentrated extracts are derivatized by trimethylsilylation with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (Nau et al., 1981; Tatsuhara et aE., 1987) or N,O-bis(trimethylsily1) trifluoroacetamide (BSTFA) (Rettenmeier et al., 19891, t-butyldimethylsilylation (Abbot et at., 1986), pentafluorobenzylation (Kassahun et al., 1989) or a combination of trimethylsilylation and pentafluorobenzylation (Kassahun et al., 1990). The original packed gas chromatography column has been substituted by capillary columns of the DB-1701 or DB-1 type. Selected ion monitoring (STM) of appropriate ions and ratios of peak heights or areas relative to various Table 2. Calibrationcurve parameters for the analysis of VPA and its metabolites Compound
Concentration range (pglmLt
Slope
Intercept
VPA 2-En (€) 2-En (2) 3-En 4-En 2,3‘-Dien 2,4-Dien 4,4‘-Dien 3-OH 4-OH 5-OH 3-Keto 3-KetoQ‘-en 4-Keto PGA
1-120 0.03-30 0.03-30 0.03-30 0.01-10 0.05-20 0.01-10 0.01-10 0.03-6 0.02-1 0 0.04-8 0.03-30 0.01-2 0.06-10 0.03- 10
0.094 0.084 0.020 0.032 0.057 0.021 0.015 0.055 0.016 0.060 0.1 12 0.069 0.041 0.027 0.067
-0.009 0.0002 0.00007 0.0002 0.0001 - 0.0003 0.0001 0.0004 -0.001 0.003 -0.002 0.046 0.00004 0.001 0.001
Correlation coefficient
0.998
> 0.999 > 0.999
0.999 0.998 > 0.999 > 0.999 0.999 0.994 > 0.999 > 0.999 0.999 0.999 0.998 0.997
E. FISHER. W. WI’ITFOHT AND H. NAU
26 ~
Table 3. Recoveries of VPA and its metabolites from serum and urine Amount added (IrglmLJ
Recovery* in serum d m L % RSD~
Amount added (pglmL)
Recoven/ in urine IrglmL
%
RSD
VPA 48.07 46.66 97.1 2.51 71.15 69.34 97.5 4.09 2-En (E) 5.52 5.34 96.7 2.96 3.47 3.41 98.3 3.43 2-En (il 0.521 0.506 97.1 5.34 2.27 2.28 100.4 2.15 3-En 1.36 1.35 99.3 4.84 2.98 2.88 96.6 6.60 4-En 0.552 0.544 98.6 1.84 0.302 0.301 99.7 1.61 2,3’-Dien 4.20 4.09 97.4 5.70 5.71 5.64 98.8 4.12 2,4-Dien 3.98 4.04 101.5 3.71 5.53 5.41 97.8 4.62 4,4’-Dien 0.263 0.255 97.0 3.92 0.251 0.244 97.2 4.60 0.539 0.507 94.1 6.42 3-OH 5.14 4.88 95.0 5.68 4-OH 0.322 0.309 96.0 5.18 3.24 2.98 92.0 4.88 5-OH 0.310 0.297 95.8 3.16 2.92 2.90 99.3 5.03 3-Keto 3.05 2.98 97.7 4.06 2.94 2.81 95.6 5.93 3-Keto-4‘-en 0.509 0.515 101.2 6.72 5.1 1 5.04 98.6 4.66 4-Keto 0.430 0.417 97.0 4.36 4.35 4.17 95.9 3.60 0.345 0.333 96.5 7.36 PGA 3.39 3.47 102.4 4.30 a Means determined from consecutive analyses of 6 aliquots of spiked blank samples. Relative standard deviation (%).
Table 4. Steady-state profiles of VPA and its metabolites in plasma and urine of 38 paediatric patients. Dose=59.79&26.51 mglkglday. Data are mean k S1) Serum
Urinea %VPA
pdmL
Wmg
% Total
VPA 78.95i26.51 644.76 f 296.18 46.89 5 13.57 2-En (E) 4.27 i 4.95 5.552 2.29 1.77 f 1.53 26.57 i23.66 2-En (Z) 0.26 f 0.16 0.335 0.20 22.40 f 14.54 1.68 f 1.OO 3-OH 0.16f0.09 0.21 20.13 49.08 5 37.40 3.68 f 1.97 4.03f2.15 5.50k3.01 3-Keto 323.27 5 220.31 22.04k 8.02 3-En 1.47 k 0.78 2.01 f 1.09 2.1 1 k 3.67 0.08 f0.1 1 2,3’-Dien 5.89 f 4.03 4.67 5 3.83 20.16f19.62 1.44f1.11 4-En 0.23 f0.14 0.29 f 0.1 9 0.99 f 1.50 0.06 0.09 3-Keto-4I-en 0.06 f0.05 0.08 ? 0.06 0.25 f0.37 4.02 t3.04 2.4’-Dien 0.97 0.41 1.42 f 0.85 3.91 f3.60 0.23 0.18 4-OH 0.50 f 0.25 0.40 f0.23 81.57f75.22 5.18k2.90 4-Keto 0.35 t 0.22 0.48 f 0.33 4.42 f 2.26 73.99 f 53.55 5-OH 0.28 f0.20 0.35 f0.25 58.26 t 48.86 3.99 f 2.86 PGA 0.1 8 f 0.1 3 0.24k 0.20 105.09f 96.12 6.8624.97 a Data given as pglmg creatinine and YOof total excreted for urine collected over 6 h after morning dose.
* *
*
Table 5. Selected patients with unusual metabolic profiles of VPA and its metabolites in plasma and urine Patient 1 Serum ILglrnL
% VPA
Urinea pglmg %Total
Patient 2 Serum Urines pglmL % VPA pglmg % Total
100.25 872.46 33.68 VPA 52.52 421.81 2-En ( E ) 17.05b 17.01 145.45 5.62 0.47 0.89 0.16 2-En 0.76 27.22 1.05 0.76 n.d. n.d. 3-OH 0.18 0.18 130.20 5.03 n.d. 0.30 3-Keto 3.92 3.91 609.76 23.54 0.90 1.71 42.50 3-En 4.47 6.26 0.24 4.46 0.81 1.54 0.26 2.3’-Dien 28.44 28.37 386.19 14.91 0.38 0.72 n.d. 4-En 0.08 0.92 0.04 0.08 0.80 1.52 1.88 n.d. 3.19 0.12 3-Keto4-en 0.07 0.13 0.65 2,4-Dien 1.28 1.28 13.40 0.52 1.98 3.77 n.d. 4-OH 0.58 107.65 4.16 0.58 0.57 1.09 15.10 4-Keto 0.43 0.43 48.93 1.89 0.54 1.03 16.93 5-OH 0.45 64.30 2.48 0.45 PGA 0.18 0.18 174.26 6.73 0.27 0.51 55.70 a Data given as pg/mg creatinine and % of total excreted for urine collected over 6 morning dose. Unusual concentrations are highlighted in bold print.
(a
75.84 0.03
-
0.05 7.64 0.05
-
0.34 0.12
-
2.71 3.04 10.01 h after
G U M S OF VALPROIC ACID AND ITS METABOLITES
internal standards were used to obtain quantitative results; this aspect of the analysis readily renders itself to automation with modern instruments.
2
m/z 199
27
We now report our own procedure suitable for the quantitative analysis of VPA and 14 metabolites in both plasma and urine. The assay presented is based on the original procedure published in 1981 (Nau et al., 1981), but allows the analysis of a larger number of metabolites by the use of Megabore columns. These columns have a lower separation efficiency compared to conventional capillary columns, but have advantages with regard to the ease of operation and accommodation of samples with large dynamic concentration ranges. This procedure has been applied to the analysis of several thousand plasma and urine samples during the past few years. Results of metabolic profiles of VPA in plasma and urine of epileptic children are presented and several abnormal metabolic patterns are identified.
EXPERIMENTAL Chemicals and reagents. The internal standard used (2-ethyl2-methylcaproic acid) was obtained from Fluka (Neu-Ulm, Germany), acetonitrile and pyridine ("dried") were from Merck (Darmstadt, Germany), ethyl acetate (nanograde) was from Promochcm (Wesel, Germany), MSTFA was from Pierce (Giinter Karl OHG, Geisenheim, Germany) and /3glucmonidase-arylsulphatase and urease were from Boehringer (Mannheim, Germany). The various VPA metabolites were donated by Drs. H. Schiifer (Desitin Werke, Hamburg, Germany), T. Baillie (Seattle, WA, USA) and B. Abbot (Vancouver, B.C., Canada). 4-en was synthesized by R.-S. Hauck (Hauck and Nau, 1989).
50
2 50
150
I
scan #
2 min
1
0
Figure 2. Representative chromatograms of urine samples from paediatric patients treated with VPA. (A) Chromatogram showing typical levels of VPA and metabolites in urine. (B) Chromatogram showing unusual pattern of VPA metabolism: high levels of 2-en, 3-en and 2,3'-dien (steady state therapy). (C) Chromatogram showing unusual pattern of VPA metabolism: high level of 4-en, low levels of other metabolites (6 h after initial VPA dose). The following concentrations were present in
these three samples: VPA
2-En ( E ) 2.3'-Dien 3-OH
4-OH 4-Keto 3-Keto 5-OH PGA
3-Keto-4I-en 4-En 3-En 2-En (Z) 2,4'-dien
A
203.2
6.08
25.88 14.03 14.62 13.18 131.56 7.37 5.08 0.61
n.d.
n.d. 3.77
3.59
B
200.8 33.81 167.06 21.89 26.91 11.19 147.25 17.17
24.90 0.79 0.19 1.66 5.96 5.79
C
454.22 0.14 n.d. n.d. n.d. n.d. 48.41 20.09 33.28 0.58 1.60 n.d. 0.04 n.d.
Hydrolysis of conjugated metabolites and removal of urea in urine samples. Urine samples (20-100 pL) were diluted to 200 pL with distilled water in a 1.5 mL Eppendorf microtube to which SO pL 1N NaH,PO, (adjusted to pH S.O), 30 pL P-glucuronidase-arylsulphatase (5 UlmL) and 10 pL urease S (900 U/mL) were added. This mixture was slowly agitated at 37 "C for 1 h and then extracted as described below. The pH was varied during treatment with @-glucuronidase between 2.5 and 8.5 in 0.5 unit steps. A broad maximum of hydrolysis was found for VPA and a number of metabolites between pH 4 and 7. Hydrolysis was therefore performed at pH 5.0, the optimal pH with regard to stability of metabolites and extraction yield. Extraction and analysis. The plasma samples (10-200 pL, depending on the concentration of VPA and on sample availability) were diluted to 200 pL with distilled water in Eppendorf 1.5 mL microvessels if necessary. To these samples, as well as to the treated urine samples (see above), 50pL 1N NaH2P04 buffer (adjusted to pH 5.0) and 1 mL ethyl acetate. containing 0.5 pg of the internal standard, were added. The samples were shaken for 20 min, centrifuged in a Model 5012 Eppendorf centrifuge for 2 min and 800 pL of the supernatant was transferred to a 1 m L glass reaction vial. After a preconcentration to about 200 pL under a stream of nitrogen 100 pL acetonitrile was added. The extraction was repeated with 1 m L ethyl acetate. The combined extracts were concentated under a stream of nitrogen to about 20 pL. Pyridine (30 pL) and MSTFA (30 pL) were then added and the vials were closed with Teflon-lined crimp tops. After standing at room temperature for several hours, or overnight, aliquots of 1 pL were injected splitless into a G U M S system (Perkin-Elmer F-22 gas chromatograph, coupled via a Jet-Separator to a Finnigan MAT CH-7A mass spectrometer
28
E. FISHER, W . WITTFOHT AND H. N A U
operated by a 2100D Superincos). The derivatives of VPA and all the metabolites were separated in a single, temperature-programmed run and quantitated by MS. The GC separations were achieved using a 30 m X 0.53 mm i.d. bonded phase Megabore DB 1701 (1.O micron film thickness) fused silica capillary column (Carlo Erba Instruments, Hofheim, Germany) with helium as carrier gas (20 rnL/min). The initial temperature of 100 "C was held for 2 min, then raised by 20 "C/niin to 250 "C. The injector temperature was 200 "C.The mass spectrometer (electron impact, 70 eV elecLron energy) was operated in the multi-ion detection mode. After a delay of 3 min (no peaks of interest) three or four ions were monitored simultaneously in three consecutive groups (Table 1). Quantitation. Standard samples were prepared by spiking drug-free human serum or urine with the highest concentrations of VPA (120 pg/mL) and metabolites (20 pg/mL). Lower concentrations were then prepared by consecutive 1:3 dilution of this highest concentration sample with serum or urine. All samples were then processed as described above. Calibration graphs were obtained by plotting the peak height ratios (VPA metabolites with respect to internal standard) versus concentrations added.
RESULTS AND DISCUSSION
Although several procedures for acidic extraction have been described (Rettenmeier et al., 1989; Tatsuhara et al., 1987; Abbott et al., 1986, Kassahun et aE., 1989; 1990), we have found extraction and derivatization under mild conditions preferable. Under acidic conditions 4-OH and 5-OH convert to their y- and 6lactones, respectively, and 3-keto is decomposed to 3heptanone. However, it is known that P-keto acids (3keto and 3-keto-4'-en) are chemically unstable under acidic conditions, and, for 4-OH, an acidic extraction medium leads to the formation of two diastcreoisomers of both open-chain and lactone forms, the latter of which does not yield a trirnethylsilyl (TMS) derivative, resulting in four peaks and leading to difficult separation and quantification. Considering these disadvantages we have found that pH 5 is optimal for adequate extraction of all VPA metabolites with ethyl acetate and does not result in significant decomposition of metabolites. Because complete enzymatic hydrolysis of conjugates occurred within 1h at pH 5 as well, this also simplified the handling of urine samples. Addition of urease during this incubation period eliminated an interfering peak produced by urea-TMS.
SIM chromatograms of the trimethylsilylated derivatives obtained from serum spiked with authentic compounds are shown in Fig. 1. Satisfactory separation of VPA and most metabolites was accomplished with a 30 m Megabore column. Only the diastereoisomeric forms of 3-OH were not completely separated and eluted as a double peak. Calibration graphs were linear in the concentration range examined from 0.01 pg/mL to 20 pg/mL (metabolites) and from l pg/mL to 120 pg/mL (VPA), with regression coefficients exceeding 0.99 in all cases (Table 2). Inter- and intra-day coefficients of deviation were < 8% in these ranges (analysis of multiple sample aliquots, n = 10) and the serum and urine extraction yields for all substances exceeded 90% (Table 3 ) . The limits of sensitivity of our assay procedure were in the low ng/mL range (signal-to-noise ratio 2: 1) using 100 pL samples. This method has been in continuous use for five years, primarily for screening patient serum and urine for abnormal patterns of metabolism. Typical patient results are given in Table 4. The /I-oxidation products of VPA, 2-en and 3-keto, as well as other major monoand di-unsaturated metabolites, 3-en and 2,3'-dien, were the major VPA metabolites in serum. The ooxidation products, 5-OH and PGA, and the w-1 oxidation products, 4-OH and 4-keto, were quantitatively less important in serum. In addition to VPA, which was present almost entirely in the conjugated form, oxidation products represented the most abundant metabolites in urine. The unsaturated metabolites, Z- and E-2-en and -2,3'-dien, were also present in significant amounts, primarily as conjugates. Examples of unusual metabolite patterns are given in Table 5 with actual chromatograms shown in Fig. 2. One unusual pattern was found in the high P-oxidation metabolites 2-en ,3-cn and E,E-2,3'-dien which developed during the course of therapy: this pattern was associated with young age ( < 2 years), cotherapy with other antiepileptic drugs and, sometimes, abnormal liver function parameters. Another unusual pattern involved increased 4-en levels after the first dose of VPA and a further one exhibited relatively high PGA levels, resembling results obtained in cases with Reye's syndrome (Fisher el al., 1989).
Acknowledgements The support and encouragement of Drs. 11. Reilh and H. Schiifer (Desitin Werke, Hamburg, Gcnnany) and the preparation of the manuscript by S. Drinkwater are gratelully acknowledged.
REFERENCES Abbott, F., Kassam, J., Acheampong, A., Ferguson, S., Panesar, S., Burton, R., Farrell, K. and Orr, J. ,(1986). J. Chromatogr. 375, 285. Baillie, T. A. (1988). Chem. Res. Toxicol. 1, 195. Chapman, A., Keane, P. E., Meldrum, B. S., Simiand, J. and Vernieres, J. C. (1982). Prog. Neurobiol. 19, 315. Driefuss, F. E., Santini, N., Langer, D. H., Sweeney, K. P., Moline, K. A. and Menander, K. B. (1987). Neurology37, 379. Dreifuss, F. E., Langer, D. H.,Moline, K. A. and Maxwell, B. A. (1989). Neurology 39, 201. Eadie, M. J., Hooper, W. D. and Dickinson, R. G . (1988). Med. Toxicol. 3, 85.
Fisher, E., Siemes, H., Pund, R . , Wittfoht, W. and Nau, H. (1991). Epilepsia, in press. Grannemann, G. R., Wang, S. I., Kesterson. J. W. and Machinist, J. M .(1984). Hepatology 4, 1153. Hauck, R. S. and Nau, H. (1989). Toxicol. Lett. 49, 41. Kassahun, K., Burton, R. and Abbott, F. S. (1989). Biomed. Environ. Mass. Spectrorn. 18, 918. Kassahun, K.. Farrell, K., Zheng, J. and Abbott, F. (1990). J. Chromatogr. 527, 327. Kesterson, J. W., Granneman, G. R. and Machinist, J. M. (1984). Hepatology 4, 1143. Loscher, W. (1985). In Handbook of Experimental
GUMS OF VALPROIC ACID AND ITS METABOLITES Pharmacology, Vol. 74 (Frey, H. H. and Janz, D., eds.), p. 507. Springer, Berlin-Heidelberg. Loscher, W. and Nau, H. (1985). Neuropharmacology 24.427. Loscher, W., Nau. H. and Siemes, H. (1988). Epilepsia 29, 31 1. Nau, H. (1986). Teratology 33, 21. Nau. H. and Hendrickx, A. G. (1987). lSI Atlas Sci.: Pharmacol. 52. Nau, H. and Loscher, W. (1982). J. Pharmacol. Exp. Ther. 220, 654. Nau, H. and Loscher, W. (1984). Epilepsia 25 (Suppl. 1). 14.
20
Nau. H., Wittfoht, W., Schafer, H., Jakobs, C., Rating, D. and Helge, H. (1981). J. Chrornatogr. 226, 69. Rettenmeier, A,, Howald, W., Levy, R., Witek, D.,Gordon, W., Porubel, D. and Baillie, T. (1989). Biomed. fnviron. Mass Spectrorn. 18, 192. Schafer, H. and Luhrs, R. (1984). In Metabolism ofAntiepileptic Drugs (Levy, R. H., Pitlick, W. H., Eichelbaum, M. and Meier, J., eds.), p. 73. Raven Press, New York. Tatsuhara, T., Muro, H., Matsuda, Y. and Irnai. Y. (1987). J. Chrornatogr. 399, 183.
Quantitative Determination of Valproic Acid and 14 Metabolites in Serum and Urine by Gas ChromatographylMass Spectrometry E. Fisher, W. Wittfoht and H. Nau* Institute for Toxicology and Embryopharmacology, Free University Berlin, D-1000 Berlin 33, Germany
A method for the determination of the antiepileptic drug valproic acid and 14 of its metabolites in serum and urine by gas chromatography/mass spectrometry with selected ion monitoring of the trimethylsilylated derivatives has been developed. Sample preparation, including hydrolysis of VPA-conjugates and removal of urea in urine is carried out at pH 5.0 and is rapid and simple. The samples are extracted with ethyl acetate and the concentrated extracts are trimethylsilylated. Analysis with adequate separation of metabolites is achieved with a DB 1701 fused silica (Megabore) capillary column. The method exhibits high recovery and reproducibility and is sufficiently sensitive and selective for analysis of small sample volumes. Application of the method for screening patient serum and urine samples for unusual metabolite patterns, with possible predictive value for early detection of liver injury, is presented.
INTRODUCTION
Valproic acid (VPA) is a widely used antiepileptic agent with proven clinical efficacy for a variety of seizure types (Chapman et al., 1982). VPA is metabolized by a number of enzymes present in microsomes, mitochondria, peroxisomes and cytoplasma (Nau and Loscher, 1982, 1984). Some metabolites, most notably the unsaturated metabolites, including 2-en-VPA and 2,3 '-dien-VPA (see Scheme 1 for abbreviations), possess anticonvulsant activity in several animal models and may therefore contribute to the therapeutic effect of VPA (Liischer and Nau, 1985; Loscher et al., 1988); such compounds may also prove to be useful antiepileptic agents by themselves (Nau, 1986; Nau and Hendrickx, 1987). A potential for hepatic failure in patients maintained * Author to whom correspondence should be addressed.
on VPA, however, especially among a recently identified high-risk group, has made hepatotoxicity associated with VPA administration a major concern (Dreifuss et al., 1987; 1989). The mechanism of VPA-induced toxicity is still unknown, but a series of clinical and experimental findings have focused attention on the extensive hepatic metabolism of VPA (Eadie et al., 1988). Several lines of investigation indicate that idiosyncratic hepatotoxicity associated with VPA administration may occur secondarily to the formation of toxic metabolites in susceptible individuals (Grannemann et al., 1984; Kesterson et a l . , 1984; Baillie, 1988; Schafer and Luhrs, 1984). Monitoring the pattern of VPA metabolism during the course of therapy may therefore help to identify those patients in whom altered metabolism of VPA presents the risk of adverse effects. Due to the large number of metabolites of VPA and their structural sifnilarity (Scheme 1), a very specific assay is required for their simultaneous determination,
on
J
3-OH
a- alucuronkle (urine)
\
f
COOH
-
valproic acid(=)
\
3-keto COOH
2.4 -dm(E)
4-en -
4 - keto -
PGA -
Scheme 1. Structures and abbreviations of VPA and metabolites measured.
0269-3879/92/010024-06 $05.00 01992 by John Wiley & Sons, Ltd.
Received 8 January 1991 Accepted 1 March 1991
GC‘/MS OF VALPROIC ACID AND ITS METABOLITES
5-0q
9.41
2s
I
mle 199
mle 185 14-e“1
I/
13-
1
4-keto
miel95
3-keto-&en
mie 215
\
044
128
212
256
TIME(MIN)
340
044
424
128
212
256
340
424
TIME(MIN)
Figure 1. Selected ion chrornatograrns for G U M S analysis of untreated patient serum spiked with VPA and metabolites.
particularly because of the large dynamic range of concentrations present: VPA plasma levels are sometimes in excess of 100 pg/mL, whereas the concentrations of some metabolites can be below 100 ng/mL. Gas chromatography/mass spectrometry (GUMS) is therefore the chosen method because not only a host of VPA metabolites must be assayed, but these compounds must also be differentiated from endogenous compounds with related structures. Since our original GC/MS method was published in 1981 (Nau et al., 1981), a number of modified procedures have been developed (Rettenmeier et al., 1989; Tatsuhara et af., 1987; Abbott et al., 1986, Kassahun et d., 1989, 1990). All procedures appear to use our ethyl acetate extraction, although we adjust the pH of plasma and urine samples to 5.0 in order to avoid formation of multiple products of some hydroxy and keto metabolites; consequently, problems arise with these metaboTable 1 . Groups of ions selected to monitor characteristic fragments of trimethylsilylated VPA and various metabolites Scan
miz
Metabolites
1-300
197 199 201 215
4,4’-dien,2,4-dien,2,3’-dien 4-en,3-en; Z- and f-2-en VPA IS”
301-420
191 199 215
3-OH 4-OH 4-keto
183 3-keto 185 S-OH, PGA 195 3-keto-4’-en IS = internal standard (2-ethyl-2-methylhexanoic acid).
421-650 a
lites when the samples are acidified to lower pH values, as pointed out by others (Rettenmeier at al., 1989). The concentrated extracts are derivatized by trimethylsilylation with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (Nau et al., 1981; Tatsuhara et aE., 1987) or N,O-bis(trimethylsily1) trifluoroacetamide (BSTFA) (Rettenmeier et al., 19891, t-butyldimethylsilylation (Abbot et at., 1986), pentafluorobenzylation (Kassahun et al., 1989) or a combination of trimethylsilylation and pentafluorobenzylation (Kassahun et al., 1990). The original packed gas chromatography column has been substituted by capillary columns of the DB-1701 or DB-1 type. Selected ion monitoring (STM) of appropriate ions and ratios of peak heights or areas relative to various Table 2. Calibrationcurve parameters for the analysis of VPA and its metabolites Compound
Concentration range (pglmLt
Slope
Intercept
VPA 2-En (€) 2-En (2) 3-En 4-En 2,3‘-Dien 2,4-Dien 4,4‘-Dien 3-OH 4-OH 5-OH 3-Keto 3-KetoQ‘-en 4-Keto PGA
1-120 0.03-30 0.03-30 0.03-30 0.01-10 0.05-20 0.01-10 0.01-10 0.03-6 0.02-1 0 0.04-8 0.03-30 0.01-2 0.06-10 0.03- 10
0.094 0.084 0.020 0.032 0.057 0.021 0.015 0.055 0.016 0.060 0.1 12 0.069 0.041 0.027 0.067
-0.009 0.0002 0.00007 0.0002 0.0001 - 0.0003 0.0001 0.0004 -0.001 0.003 -0.002 0.046 0.00004 0.001 0.001
Correlation coefficient
0.998
> 0.999 > 0.999
0.999 0.998 > 0.999 > 0.999 0.999 0.994 > 0.999 > 0.999 0.999 0.999 0.998 0.997
E. FISHER. W. WI’ITFOHT AND H. NAU
26 ~
Table 3. Recoveries of VPA and its metabolites from serum and urine Amount added (IrglmLJ
Recovery* in serum d m L % RSD~
Amount added (pglmL)
Recoven/ in urine IrglmL
%
RSD
VPA 48.07 46.66 97.1 2.51 71.15 69.34 97.5 4.09 2-En (E) 5.52 5.34 96.7 2.96 3.47 3.41 98.3 3.43 2-En (il 0.521 0.506 97.1 5.34 2.27 2.28 100.4 2.15 3-En 1.36 1.35 99.3 4.84 2.98 2.88 96.6 6.60 4-En 0.552 0.544 98.6 1.84 0.302 0.301 99.7 1.61 2,3’-Dien 4.20 4.09 97.4 5.70 5.71 5.64 98.8 4.12 2,4-Dien 3.98 4.04 101.5 3.71 5.53 5.41 97.8 4.62 4,4’-Dien 0.263 0.255 97.0 3.92 0.251 0.244 97.2 4.60 0.539 0.507 94.1 6.42 3-OH 5.14 4.88 95.0 5.68 4-OH 0.322 0.309 96.0 5.18 3.24 2.98 92.0 4.88 5-OH 0.310 0.297 95.8 3.16 2.92 2.90 99.3 5.03 3-Keto 3.05 2.98 97.7 4.06 2.94 2.81 95.6 5.93 3-Keto-4‘-en 0.509 0.515 101.2 6.72 5.1 1 5.04 98.6 4.66 4-Keto 0.430 0.417 97.0 4.36 4.35 4.17 95.9 3.60 0.345 0.333 96.5 7.36 PGA 3.39 3.47 102.4 4.30 a Means determined from consecutive analyses of 6 aliquots of spiked blank samples. Relative standard deviation (%).
Table 4. Steady-state profiles of VPA and its metabolites in plasma and urine of 38 paediatric patients. Dose=59.79&26.51 mglkglday. Data are mean k S1) Serum
Urinea %VPA
pdmL
Wmg
% Total
VPA 78.95i26.51 644.76 f 296.18 46.89 5 13.57 2-En (E) 4.27 i 4.95 5.552 2.29 1.77 f 1.53 26.57 i23.66 2-En (Z) 0.26 f 0.16 0.335 0.20 22.40 f 14.54 1.68 f 1.OO 3-OH 0.16f0.09 0.21 20.13 49.08 5 37.40 3.68 f 1.97 4.03f2.15 5.50k3.01 3-Keto 323.27 5 220.31 22.04k 8.02 3-En 1.47 k 0.78 2.01 f 1.09 2.1 1 k 3.67 0.08 f0.1 1 2,3’-Dien 5.89 f 4.03 4.67 5 3.83 20.16f19.62 1.44f1.11 4-En 0.23 f0.14 0.29 f 0.1 9 0.99 f 1.50 0.06 0.09 3-Keto-4I-en 0.06 f0.05 0.08 ? 0.06 0.25 f0.37 4.02 t3.04 2.4’-Dien 0.97 0.41 1.42 f 0.85 3.91 f3.60 0.23 0.18 4-OH 0.50 f 0.25 0.40 f0.23 81.57f75.22 5.18k2.90 4-Keto 0.35 t 0.22 0.48 f 0.33 4.42 f 2.26 73.99 f 53.55 5-OH 0.28 f0.20 0.35 f0.25 58.26 t 48.86 3.99 f 2.86 PGA 0.1 8 f 0.1 3 0.24k 0.20 105.09f 96.12 6.8624.97 a Data given as pglmg creatinine and YOof total excreted for urine collected over 6 h after morning dose.
* *
*
Table 5. Selected patients with unusual metabolic profiles of VPA and its metabolites in plasma and urine Patient 1 Serum ILglrnL
% VPA
Urinea pglmg %Total
Patient 2 Serum Urines pglmL % VPA pglmg % Total
100.25 872.46 33.68 VPA 52.52 421.81 2-En ( E ) 17.05b 17.01 145.45 5.62 0.47 0.89 0.16 2-En 0.76 27.22 1.05 0.76 n.d. n.d. 3-OH 0.18 0.18 130.20 5.03 n.d. 0.30 3-Keto 3.92 3.91 609.76 23.54 0.90 1.71 42.50 3-En 4.47 6.26 0.24 4.46 0.81 1.54 0.26 2.3’-Dien 28.44 28.37 386.19 14.91 0.38 0.72 n.d. 4-En 0.08 0.92 0.04 0.08 0.80 1.52 1.88 n.d. 3.19 0.12 3-Keto4-en 0.07 0.13 0.65 2,4-Dien 1.28 1.28 13.40 0.52 1.98 3.77 n.d. 4-OH 0.58 107.65 4.16 0.58 0.57 1.09 15.10 4-Keto 0.43 0.43 48.93 1.89 0.54 1.03 16.93 5-OH 0.45 64.30 2.48 0.45 PGA 0.18 0.18 174.26 6.73 0.27 0.51 55.70 a Data given as pg/mg creatinine and % of total excreted for urine collected over 6 morning dose. Unusual concentrations are highlighted in bold print.
(a
75.84 0.03
-
0.05 7.64 0.05
-
0.34 0.12
-
2.71 3.04 10.01 h after
G U M S OF VALPROIC ACID AND ITS METABOLITES
internal standards were used to obtain quantitative results; this aspect of the analysis readily renders itself to automation with modern instruments.
2
m/z 199
27
We now report our own procedure suitable for the quantitative analysis of VPA and 14 metabolites in both plasma and urine. The assay presented is based on the original procedure published in 1981 (Nau et al., 1981), but allows the analysis of a larger number of metabolites by the use of Megabore columns. These columns have a lower separation efficiency compared to conventional capillary columns, but have advantages with regard to the ease of operation and accommodation of samples with large dynamic concentration ranges. This procedure has been applied to the analysis of several thousand plasma and urine samples during the past few years. Results of metabolic profiles of VPA in plasma and urine of epileptic children are presented and several abnormal metabolic patterns are identified.
EXPERIMENTAL Chemicals and reagents. The internal standard used (2-ethyl2-methylcaproic acid) was obtained from Fluka (Neu-Ulm, Germany), acetonitrile and pyridine ("dried") were from Merck (Darmstadt, Germany), ethyl acetate (nanograde) was from Promochcm (Wesel, Germany), MSTFA was from Pierce (Giinter Karl OHG, Geisenheim, Germany) and /3glucmonidase-arylsulphatase and urease were from Boehringer (Mannheim, Germany). The various VPA metabolites were donated by Drs. H. Schiifer (Desitin Werke, Hamburg, Germany), T. Baillie (Seattle, WA, USA) and B. Abbot (Vancouver, B.C., Canada). 4-en was synthesized by R.-S. Hauck (Hauck and Nau, 1989).
50
2 50
150
I
scan #
2 min
1
0
Figure 2. Representative chromatograms of urine samples from paediatric patients treated with VPA. (A) Chromatogram showing typical levels of VPA and metabolites in urine. (B) Chromatogram showing unusual pattern of VPA metabolism: high levels of 2-en, 3-en and 2,3'-dien (steady state therapy). (C) Chromatogram showing unusual pattern of VPA metabolism: high level of 4-en, low levels of other metabolites (6 h after initial VPA dose). The following concentrations were present in
these three samples: VPA
2-En ( E ) 2.3'-Dien 3-OH
4-OH 4-Keto 3-Keto 5-OH PGA
3-Keto-4I-en 4-En 3-En 2-En (Z) 2,4'-dien
A
203.2
6.08
25.88 14.03 14.62 13.18 131.56 7.37 5.08 0.61
n.d.
n.d. 3.77
3.59
B
200.8 33.81 167.06 21.89 26.91 11.19 147.25 17.17
24.90 0.79 0.19 1.66 5.96 5.79
C
454.22 0.14 n.d. n.d. n.d. n.d. 48.41 20.09 33.28 0.58 1.60 n.d. 0.04 n.d.
Hydrolysis of conjugated metabolites and removal of urea in urine samples. Urine samples (20-100 pL) were diluted to 200 pL with distilled water in a 1.5 mL Eppendorf microtube to which SO pL 1N NaH,PO, (adjusted to pH S.O), 30 pL P-glucuronidase-arylsulphatase (5 UlmL) and 10 pL urease S (900 U/mL) were added. This mixture was slowly agitated at 37 "C for 1 h and then extracted as described below. The pH was varied during treatment with @-glucuronidase between 2.5 and 8.5 in 0.5 unit steps. A broad maximum of hydrolysis was found for VPA and a number of metabolites between pH 4 and 7. Hydrolysis was therefore performed at pH 5.0, the optimal pH with regard to stability of metabolites and extraction yield. Extraction and analysis. The plasma samples (10-200 pL, depending on the concentration of VPA and on sample availability) were diluted to 200 pL with distilled water in Eppendorf 1.5 mL microvessels if necessary. To these samples, as well as to the treated urine samples (see above), 50pL 1N NaH2P04 buffer (adjusted to pH 5.0) and 1 mL ethyl acetate. containing 0.5 pg of the internal standard, were added. The samples were shaken for 20 min, centrifuged in a Model 5012 Eppendorf centrifuge for 2 min and 800 pL of the supernatant was transferred to a 1 m L glass reaction vial. After a preconcentration to about 200 pL under a stream of nitrogen 100 pL acetonitrile was added. The extraction was repeated with 1 m L ethyl acetate. The combined extracts were concentated under a stream of nitrogen to about 20 pL. Pyridine (30 pL) and MSTFA (30 pL) were then added and the vials were closed with Teflon-lined crimp tops. After standing at room temperature for several hours, or overnight, aliquots of 1 pL were injected splitless into a G U M S system (Perkin-Elmer F-22 gas chromatograph, coupled via a Jet-Separator to a Finnigan MAT CH-7A mass spectrometer
28
E. FISHER, W . WITTFOHT AND H. N A U
operated by a 2100D Superincos). The derivatives of VPA and all the metabolites were separated in a single, temperature-programmed run and quantitated by MS. The GC separations were achieved using a 30 m X 0.53 mm i.d. bonded phase Megabore DB 1701 (1.O micron film thickness) fused silica capillary column (Carlo Erba Instruments, Hofheim, Germany) with helium as carrier gas (20 rnL/min). The initial temperature of 100 "C was held for 2 min, then raised by 20 "C/niin to 250 "C. The injector temperature was 200 "C.The mass spectrometer (electron impact, 70 eV elecLron energy) was operated in the multi-ion detection mode. After a delay of 3 min (no peaks of interest) three or four ions were monitored simultaneously in three consecutive groups (Table 1). Quantitation. Standard samples were prepared by spiking drug-free human serum or urine with the highest concentrations of VPA (120 pg/mL) and metabolites (20 pg/mL). Lower concentrations were then prepared by consecutive 1:3 dilution of this highest concentration sample with serum or urine. All samples were then processed as described above. Calibration graphs were obtained by plotting the peak height ratios (VPA metabolites with respect to internal standard) versus concentrations added.
RESULTS AND DISCUSSION
Although several procedures for acidic extraction have been described (Rettenmeier et al., 1989; Tatsuhara et al., 1987; Abbott et al., 1986, Kassahun et aE., 1989; 1990), we have found extraction and derivatization under mild conditions preferable. Under acidic conditions 4-OH and 5-OH convert to their y- and 6lactones, respectively, and 3-keto is decomposed to 3heptanone. However, it is known that P-keto acids (3keto and 3-keto-4'-en) are chemically unstable under acidic conditions, and, for 4-OH, an acidic extraction medium leads to the formation of two diastcreoisomers of both open-chain and lactone forms, the latter of which does not yield a trirnethylsilyl (TMS) derivative, resulting in four peaks and leading to difficult separation and quantification. Considering these disadvantages we have found that pH 5 is optimal for adequate extraction of all VPA metabolites with ethyl acetate and does not result in significant decomposition of metabolites. Because complete enzymatic hydrolysis of conjugates occurred within 1h at pH 5 as well, this also simplified the handling of urine samples. Addition of urease during this incubation period eliminated an interfering peak produced by urea-TMS.
SIM chromatograms of the trimethylsilylated derivatives obtained from serum spiked with authentic compounds are shown in Fig. 1. Satisfactory separation of VPA and most metabolites was accomplished with a 30 m Megabore column. Only the diastereoisomeric forms of 3-OH were not completely separated and eluted as a double peak. Calibration graphs were linear in the concentration range examined from 0.01 pg/mL to 20 pg/mL (metabolites) and from l pg/mL to 120 pg/mL (VPA), with regression coefficients exceeding 0.99 in all cases (Table 2). Inter- and intra-day coefficients of deviation were < 8% in these ranges (analysis of multiple sample aliquots, n = 10) and the serum and urine extraction yields for all substances exceeded 90% (Table 3 ) . The limits of sensitivity of our assay procedure were in the low ng/mL range (signal-to-noise ratio 2: 1) using 100 pL samples. This method has been in continuous use for five years, primarily for screening patient serum and urine for abnormal patterns of metabolism. Typical patient results are given in Table 4. The /I-oxidation products of VPA, 2-en and 3-keto, as well as other major monoand di-unsaturated metabolites, 3-en and 2,3'-dien, were the major VPA metabolites in serum. The ooxidation products, 5-OH and PGA, and the w-1 oxidation products, 4-OH and 4-keto, were quantitatively less important in serum. In addition to VPA, which was present almost entirely in the conjugated form, oxidation products represented the most abundant metabolites in urine. The unsaturated metabolites, Z- and E-2-en and -2,3'-dien, were also present in significant amounts, primarily as conjugates. Examples of unusual metabolite patterns are given in Table 5 with actual chromatograms shown in Fig. 2. One unusual pattern was found in the high P-oxidation metabolites 2-en ,3-cn and E,E-2,3'-dien which developed during the course of therapy: this pattern was associated with young age ( < 2 years), cotherapy with other antiepileptic drugs and, sometimes, abnormal liver function parameters. Another unusual pattern involved increased 4-en levels after the first dose of VPA and a further one exhibited relatively high PGA levels, resembling results obtained in cases with Reye's syndrome (Fisher el al., 1989).
Acknowledgements The support and encouragement of Drs. 11. Reilh and H. Schiifer (Desitin Werke, Hamburg, Gcnnany) and the preparation of the manuscript by S. Drinkwater are gratelully acknowledged.
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Fisher, E., Siemes, H., Pund, R . , Wittfoht, W. and Nau, H. (1991). Epilepsia, in press. Grannemann, G. R., Wang, S. I., Kesterson. J. W. and Machinist, J. M .(1984). Hepatology 4, 1153. Hauck, R. S. and Nau, H. (1989). Toxicol. Lett. 49, 41. Kassahun, K., Burton, R. and Abbott, F. S. (1989). Biomed. Environ. Mass. Spectrorn. 18, 918. Kassahun, K.. Farrell, K., Zheng, J. and Abbott, F. (1990). J. Chromatogr. 527, 327. Kesterson, J. W., Granneman, G. R. and Machinist, J. M. (1984). Hepatology 4, 1143. Loscher, W. (1985). In Handbook of Experimental
GUMS OF VALPROIC ACID AND ITS METABOLITES Pharmacology, Vol. 74 (Frey, H. H. and Janz, D., eds.), p. 507. Springer, Berlin-Heidelberg. Loscher, W. and Nau, H. (1985). Neuropharmacology 24.427. Loscher, W., Nau. H. and Siemes, H. (1988). Epilepsia 29, 31 1. Nau, H. (1986). Teratology 33, 21. Nau. H. and Hendrickx, A. G. (1987). lSI Atlas Sci.: Pharmacol. 52. Nau, H. and Loscher, W. (1982). J. Pharmacol. Exp. Ther. 220, 654. Nau, H. and Loscher, W. (1984). Epilepsia 25 (Suppl. 1). 14.
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Nau. H., Wittfoht, W., Schafer, H., Jakobs, C., Rating, D. and Helge, H. (1981). J. Chrornatogr. 226, 69. Rettenmeier, A,, Howald, W., Levy, R., Witek, D.,Gordon, W., Porubel, D. and Baillie, T. (1989). Biomed. fnviron. Mass Spectrorn. 18, 192. Schafer, H. and Luhrs, R. (1984). In Metabolism ofAntiepileptic Drugs (Levy, R. H., Pitlick, W. H., Eichelbaum, M. and Meier, J., eds.), p. 73. Raven Press, New York. Tatsuhara, T., Muro, H., Matsuda, Y. and Irnai. Y. (1987). J. Chrornatogr. 399, 183.
