BIOMEDICAL CHROMATOGRAPHY, VOL. 5,68-73 (1991)
Simultaneous Determination of Gemfibrozil and its Metabolites in Plasma and Urine by a Fully Automated High Performance Liquid Chromatographic System Akihiko Nakagawa", Akemi Shigeta, Haruo Iwabuchi, Masaaki Horiguchi?, Kan-ichi Nakamura and Hidekuni Takahagi Analytical and Metabolic Research Laboratories. Sankyo Co. Ltd., 2-58, Hiromachi 1-chome. Shinagawa-ku, Tokyo 140, Japan
Sensitive and specific methods for the simultaneous determination of gemfibrozil (Lopid@),a lipid-lowering agent, and its metabolites in plasma and urine are described. The methods are based on a fully automated high performance liquid chromatographic (HPLC) system with fluorescence detection. Urine samples, diluted with acetonitrile, were directly analysed by HPLC using a flow and eluent programming method. In the case of plasma, gemfibruzil and its main metabolites were extracted from acidified samples and the resulting extracts injected into the chromatographic system. The sensitivity was approximately 100 ng/mL for gemfibrozil and its four metabolites using 0.5 mL plasma or urine. An acyl glucuronide of gemfibrozil excreted in human urine after oral administration of the drug was isolated and its structure and stability examined.
INTRODUCTION Gemfibrozil, S-(2,S-dimethylphenoxy)-2,2-dimethylpentamic acid (Fig. l), is an oral lipid lowering agent developed by Warner-Lambert (Tuomilehto et al., 1976; Nash, 1980; Jain et al., 1981). The metabolic fate of gemfibrozil was reported by Okerholm et al. (1976) using 3H-labelled compounds. According to this, gemfibrozil is extensively metabolized and excreted in urine as a glucuronide together with four oxidized metabolites (Fig. l), the chief of which is a benzoic acid derivative, M3. For the metabolic profile of gemfibrozil in plasma, nothing has been described except the main
metabolite, M3 (Randinitis et al., 1984a). The determination of gemfibrozil only in plasma and urine can be carried out using gas chromatography (GC) (Randinitis et al., 1984b) and high performance liquid chromatography (HPLC) (Randinitis et a / . , 1984a; Hengy and Koelle, 1985; Forland et al., 1987). The present study describes the simultaneous determination of gcmfihrozil and its metabolites in human plasma and urine by HPLC. Some preliminary results on the metabolic fate of gemfibrozil in humans are also mentioned. The conjugated metabolite of gemfibrozil was elucidated after isolation from urine.
EXPERIMENTAL 73
R1 R2 R3 n __________________________ G e m f ibrozil CH3 H CH3 3 MI CH3 OH CH3 3 M2 CH2OH H CH3 3 M3 COOH H CH3 3 M4 H CHZOH 3 CH3 H CH3 2 c2 CH3 H CH3 5 c5 CH3 Figure 1. Structures of gemfibrozil and its related compounds. * Author to whom correspondence should be addressed iPrcscnt address: Institute of Science and Technology, Inc., 10-2,
Kitashinagawa _?-chome, Shinagawa-ku, Tokyo 140, Japan. 0269-3879/9 1/020M8-06 $05 .oo
01991 by John Wifey s( Sons, Ltd
Chemicals. Gemfibrozil, its four metabolites (Ml-4) and two internal standards (C2, CS) were supplied from Warner-Lambert K. K. (Tokyo, Japan). Glusulase@ was obtained from Dupont (Boston, MA, USA). All solvents and chemicals used were of IIPLC or analytical reagent grade and no further purification was carried out. Apparatus. The IIPLC system used was as follows: Two pumps (LCdA; Shirnadzu, Kyoto, Japan), an automatic sample injector (SIL-hA; Shimadzu), two fluorescence detectors (F-1000; Hitachi, Tokyo, Japan), two integrators (HP3393; Hewlett-Packard, Avondale, PA, USA) and a system controller (SCL-6A; Shimadzu). Peak area data from integrators were processed by an on-line-connected personal computer (PC-9801; NEC, Tokyo, Japan). The flow diagram of the total system employed i s shown in Fig. 2. A fluorescence spectrophotometer (650- 10s: Hitachi). a high-speed muti-wavelength spectrophotomcter (1 IP-1040A; Received 9 April IYW Accepted 14 June I990
SIMULTANEOUS DETERMINATION OF GEMFTBROZIL AND ITS METABOLITES
hY ~
Tablel. Ex and Em wavelengths of maximum fluorescence intensity for gemfibrozil and its four metabolites
-PLOY SIGNAL
Gemfibrozil MI M2
DETECTOR-2
, , INTEGRATOR-2
Figure 2. Flow diagram of a fully automated HPLC system. Thick arrows indicate the sample flow system; broken arrows indicate the operational sequence undergoing the signal.
Hewlett-Packard), and a mass spectrometer equipped with a SLMS ion source (M-80A; Hitachi) were used for qualitative analysis. Chromatographic conditions. For urine samples, the column was a YMC-A312 ODS (15 cm X 4.6 mm I.D.; Yamamura Chemicals, Kyoto, Japan) held at room temperature. The initial mobile phase, consisting of 10 mM acetate buffer (pH4.7)/acetonitrile (55:45), was driven at a flow rate of 1 mL/min. At 10.5 min after sample injection, the mobile phase was changed to a higher acetonitrile content (80%) with a 2 mLImin flow rate. The excitation (Ex) and emission (Em) wavelengths of two detectors were set at 283 and 315 nm, and 300 and 340 nm, respectively, for the simultaneous determination of the drug and its metabolites. A ChH5-1252N(25 cm x 4.6 mm i.d.; Senshu Sci., Tokyo, Japan) column was used for plasma samples at room temperature with 10 mM tartarate buffer (pH 3.3)/acetonitrile/PICBA (4852:O.S) as the eluent at a flow rate of 1mLimin. A fluorescence detector was operated at Ex:293 nm and Em:325 nm. For analysis of the conjugated forms of gernfibrozil, YMC-A312 ODS was used at room temperature with 10 mM acetate buffer (pH 4.7)lacetonitrile (64:36) as the eluent at a flow rate of 1.0 mL/min. Isolation of the conjugates was achieved using the same column with a linear gradient elution (acetonitrile, 0-45%, 30 min). Sample preparation. To assay gemfibrozil and its metabolites in urine excreted as the free form, 0.5 mL acetonitrile containing 10pg/mL C5 as the internal standard was added to 0.5mL urine. The sample was vortexed, centrifuged at 2000 g, and 10 pL of the upper layer was injected into the chromatograph. For the hydrolysis of conjugated gemfibrozil and its metabolites, 0.5 mL Clusulase@solution, diluted with 4 volumes I M acetate buffer (pfl5.2) containing 10% (w/v) sodium metabisulfite (Na2S205),was added to each 0.5mL urine sample. After reciprocal incubation at 37°C for 2 h, 3 mL acetonitrile containing 10 pg/mL C5 was added to the hydrolysate. The mixture was vortexed and centrifuged at 2000 g, and 10 pL of the upper layer was injected into the chromatograph. To the plasma samples (0.5 mL) were added 0.5 mL 1% acetonitrile/phosphate buffered saline (pH 7.4), containing C2 (10 yg/mL) as the internal standard, and 20 pL of formic acid, and the samples were then extracted with 5 mL ethyl acetate/cyclohexane (2:8). After evaporating the solvent, the resulting extracts were reconstructed with 0.5 mL of the eluent and injected to the chromatograph (10 pL).
M3 M4
Ex(nrn)
Ern(nrn)
283 295 280 300 280
315 330 308
340 308
RESULTS AND DISCUSSION Fluorescence spectra For the determination of gemfibrozil and its metabolites by HPLC, fluorescence detection was first introduced in the present study, since these compounds were found to have fluorescence derived from the phenoxy moiety. The maximum excitation (Ex) and emission (Em) wavelengths of these compounds are listed in Table 1. The differences in the wavelength in fluorescence detection in these compounds may be derived from substituents on the phenoxy ring. The detection limit of gemfibrozil was compared between fluorescence and UV detection. UV detection was reconstructed using the same HPLC conditions as described by Hengy and Koelle (1985). At 1ng injection of gemfibrozil to HPLC, the signal-to-noise ratios in the fluorescence and UV peaks were 6.5 and 5.5, respectively. However, the former method was more advantageous in its stability of chromatographic baseline and selectivity for the compounds of interest in body fluids. HPLC separation of the four metabolites of gemfibrozil
The separation of the four metabolites by octadecylsilanized silica (ODS) column was examined using acetate buffers having various pH values and containing 45% acetonitrile as the eluent. The relationships between the pH of the eluent and the capacity factor, k ' , of these metabolites are shown in Fig. 3. The k' values of three monohydroxy isomers of gemfibrozil metabolites,
-A-
M1 M2 & M3
---+-
k'
--
M4
I--
03.5
4.0
5.0
4.5
5.5
6.0
PH
Figure 3. The relationships between pH and k' values of four
metabolites. For HPLC conditions see text.
A . NAKAGAWA
70
'I'ahle 2. The resolutian value (Rs) between M1, M2. M3, and M4 pH
M2-M3
M2-M4
Ml-M4
4.4 4.7 5.0
4.25 8.47 12.83
2.66 2.46 2.12
1.03 1.30 1.70
M I , M2. and M4. were similarly decreased with increasing pHs. The degree of decrease in the k' value of the dicarboxylic acid metabolite, M3, was more steep than the other metabolites. These four metabolites could be separated by the ODS column at pH values from 4.4 to 5.0. At this pH range the resolution values, Rs, are listed in Table 2. The maximum resolution was obtained at pH 5.0. However, the k' value of M3 was so small (0.785) that 1 0 m ~acetate buffer (pH 4.7)iacetonitrile (55:45) was chosen for the elution of these metabolites. Flow programming elution
Since the k' of gemfibrozil was more than 30 under the above HPLC conditions. the eluent was programmed to increase to a higher acetonitrile content ( ~ W O and) a flow rate of 2 mL/min after the elution of these metabolites. This flow programming enabled the elution of both gemfibrozil and the internal standard, C5, within 20min. A drift of the chromatographic baseline of fluorescence detection was not observed before and after the change of eluent and flow rate. Selection of fluorescence wavelength
As shown in Table 1, gemfibrozil and its metabolites give their maximum fluorescence intensities at different wavelengths. Under the above HPLC conditions, Ex at 283 and E m at 315 nm gave the maximum intensity for gemfibrozil, but the peak responses of M1 and M3 were decreased to 80% and lo%, respectively, compared to those obtained by the optimum wavelength for the individual compounds. We solved thi5 problem by utilizing two fluorescence detectors in series. The Ex and Em wavelengths of the first detector were set for determining gemfibrozil, M2, M4, and C5, and the second one, connected in series, was set at 300 (Ex) and 340 (Em) nm for MI and M3, considering the possible effect of diffusion after eluting the column on the separation of the compounds concerned.
Kr A L
Chromatograms of human control and gemfibroziladministered urine after enzymatic hydrolysis are shown in Fig. 4. Small interfering peaks were observed at the retention times of M3 (Fig. 4C), M2 and M4 (Fig. 4A). However, urinary concentrations of these metabolites after thc oral administration of gemfibrozil a t clinical doses were so high that these pcaks did not interfere practically. The amount of liberated gemfibrozil and its four metabolites during enzymatic hydrolysis reached a maximum at 2 h after incubation. After 20 h incubation, the amount of M1 liberated was decreased slightly (ca. 10%) compared to that at 2 h incubation. The hydrolysate with Glusulase@had many interfering peaks when monitored by UV detector. and further purification steps were required for the quantitative analysis. On the other hand, fluorescence detection permitted more selective and sensitive determination than UV, because the fluorescent peaks derived from the enzyme preparation had little effect on the quantitation of gemfibrozil and its metabolites, as shown in Fig. 4. Calibration curves for gemfibrozil and its metabolites were prepared using human control urine spiked with various amounts of standard compounds. Table 3 shows the reproducibility and linearity of the present method, including the enzymatic hydrolysis, for determining the total amounts of these compounds. The values for gemfibrozil and its four metabolites gave satisfactory results for the coefficients of variation (0.999). The quantitative recoveries of these compounds may be due to there being no extraction step in the procedure. The analytical parameters for the free fraction in urine without enzymatic hydrolysis were almost the same as those obtained after enzymatic treatment. Determination of gemfibrozil, M1 and M3 in plasma
The plasma concentrations of the minor metabolites, M2 and M4, wcrc lcss than 30 ng/mL after a 450 mg dose of gemfibrozil in preliminary G U M S quantitation
Determination of gemfibrozil and its metabolites in urine
Gemfibrozil and its metabolites were excreted in urine as conjugates with glucuronic acid, except for M3, which was present mainly as the free form (Okerholm et af., 1976; Randinitis et at., 1984a). Hydrolysis of the conjugates was carried out using Glusulase@at a higher concentration than the conventional method, in the presence of sodium metabisulfite as an antioxidant, to prevent degradation of the compounds (Nakagawa et af., 1982). The hydrolysis rates were checked by the present method.
I I Ir I I . I 1 I 0 5 10 15 20 0 5 10 15 20 Time ( rnin ) Time ( min )
Figure 4. HPLC chromatograms of humar urine samples: (A) and (C)blank urine; (B) and (D) human urine after oral adrninistration of gemfibrozil (450 mglbody). (A)and (B) were detected at Ex:283 nrn and Em:315 nm; (C) and (D) were detected a t Ex:300 nrn and Em:340 nrn.
71
SIMULTANEOUS DETERMINATION OF GEMFIBROLIL AND ITS METABOLITES ~
Table 3. Reproducibility and linearity of the analyses of gemfibrozil and its metabolites in urine . ; Compound
Conc (WgimLl
Coefficient of variation
Conc ranges WmL)
Correlation coefficient
Gemfibrozil MI M2 M3 M4
135 9.7 3.3 22 9 2.9
0.810 1.421 0.668 0.969 0.974
3.00-600 0.64-64 0.15-30 1.80-300 0.15-30
0.999 0.999 0.999 0.999 0.999
"
5 \ Pi 0 1 E
t/2B
1
(hr-1) (l/hr) (hr-I)
0.603
(hr)
1 .41 0.56
Lag time (hr)
10
0.4 7 7 94 1
5
(Nakagawa ef ul., unpublished data), whereas plasma concentrations of gemfbrozil and M3 were estimated to be higher than 100 ng/mL, even 24 h after a single oral administration of the drug at clinical doses (Randinitis et ul., 1Y84a). Since the present HPLC method could not detect concentration levels of less than 100 ng/mL, the simultaneous determination of gcmfibrozil, M1, and M3 was carried out. Several types of chemically modified phases such as C,,, C,, C4, C?, CN, ChH5.and so on. were examined for suitability. The C,H, (pheny1)-bonded phase was found to be the most suitable (Fig. 5 ) . C2 (scc Fig. 1) was chosen as t h e internal standard. Under these IIPLC conditions, M2 anti M4, the two miiior metabolites. would elute between M1 and M3. Figure 5 shows chromatograms of extracts from human plasma (see Experimental Section) after oral administration of gemfihrozil (450 mg/body) when monitored under three different fluorescent conditions. Gemfihrozil, M1 and M3 could be detected with good sensitivity at a wavelength near the maximum fluorescence intensity of M1 (Fig. SA)). In addition. thcse conditions have the advantage that non-separable minor metabolites, M2 and M4, have less effect on the determination of other metabolites. Kecoveries were tested by extracting with 5 mL ethyl acetate/cyclohexanc (2:s) from acidified plasma (0.5 mL) spiked with 5 pg each of gemfibrozil, M1, M3 and C2. These compounds were efficiently extracted. with recovery ratcs of more than 95%. The reproducibility. linearity and sensitivity o f the present method
v 2 4 6
0
Time [ min
-
8
0
1
-
2
4
Time
6
1 min
8
--I 0
2
4
6
8
Time [ win
Figure 5. HPLC chromatograms using CsH, bonded phases. (A), (B) and (C):human plasma after receiving gemfibrozil; (D), ( E l and (F): control human plasma at different fluorescence wavelengths.
0 0
2
4
6
8
12
24
Time
(
hour
)
Figure 6. Plasma concentration-time curves of gemfibrozil, M I and M3 after a single oral administration of the drug (450 mg/ body, n = 6). The pharmacokinetic parameters of gemfibrozil were calculated from mean plasma concentrations.
were also examined using 0.5 mL human plasma spiked with 0.05-25 pg of these compounds. Coefficients of variation expressed as the relative standard deviation (RSD) on the determination of gemtibrozil and M1 were less than 2Y0 in the concentration range of 110 pglmL in plasma. Good correlation coefficients ( r 1 0 . 9 9 9 ) were also obtained for these two compounds in the concentration ranges tested. On the other hand, these parameters for M3 were not as good as those of gemfibrozil and M1. The RSD values for M3 were 4.4 and 4.6% at concentrations of 10 and 1 pglmL, respectively. The correlation coefficient was 0.Y98 in the conccntration range 0.1-50,ugl mL in plasma. However, the present method was still useful for the determination of M3 in plasma.
Practical application for a clinical study
The present methods were applied t o the determination of the plasma concentration and urinary excretion of gemfibrozil and its metabolites after a single oral administration of the drug to six healthy adult malc volunteers (450mg/body). This study was carried out as a part o f a Phase I trial of gemfibrozil. Figure 6 shows the mean plasma concentration-time curves of gemfihro~il,MI and M3. The pharmacokinetic parameters of gemfibrozil were calculated from the mean plasma concentration based on a onecompartment with lag-time absorption model and are listed in Fig. 6. The biological half-life of the drug (1.4 h) was in good agreement with the previously reported data (Randinitis ef af., 1984a). The plasma concentration profile of M3 showed a rather unusual pattern-a plateau at 6-12 h after administration of the drug, with a further increase at 24 h. This seemed attributable to enterohcpatic circulation of this mctabolite (Okerholm el ul., 1976; Randinitis et ul., 1984a). A detailed pharmacokinetic interpretation of M3 in humans will bc mentioned elsewhere. Urinary excretions of gemfibrozil and its metabolites are listcd in Table 4. These values are in good agreement with the data reported by Okerholm er al. (1976) using 3H-labelled gemfbrozil. except for the values obtained for M1 and M-C.
A. NAKAGAWA ETAL
72
Table 4. Determination of 0-24 h urinary gemfibrozil and its metabolites in human (% of dose, 450 mglbody) Compound
Free
Conjugate
Total
M4
0.59 2 0.13 0.08 f 0.01 0.01 f 0.00 15.34 k 2.19 0.01 k 0.00
32.09k 1.30 7.33 k 0.83 0.70 k 0.06 4.51 k0.52 0.50k 0.02
32.68 t 1.35 7.41 f 0.83 0.71 f0.06 19.85+2.18 0.51 20.06
Total
16.04k 2.17
45.13 k 1.57
61.17 k 2.71
Gemfibrozil
M1 M2 M3
Stability of the gemfibrozil conjugate
Gemfibrozil was reported as being excreted in urine as an acyl glucuronide conjugate (Okerholm et al., 1976; Randinitis et al., 1984a, 1984b). The properties of various acyl glucuronides have been reviewed by Faed (1984). According to this, acyl glucuronides are likely to undergo facile intramolecular rearrangement under neutral-to-weak basic conditions, to form 2-0-, 3-0and 4-0-acyl glucuronides. Among them, only 1-0-acyl glucuronide could be the substrate for [email protected] this regard, the properties and stability of the acyl glucuronide of gemfibrozil were examined. Urine samples adjusted to pH values of 7.0, 7.5 and 8.0, respectively, were divided into two portions. One portion was rapidly frozen at -20 "C and the other was kept at room temperature. After two days of storage the urine samples were directly injected into the HPLC system (see Experimental Section). Figure 7 shows chromatograms of these samples under optimal HPLC conditions. The upper chromatograms are those of the urine samples stored at -20°C. The most prominent peak (named as Ul), which eluted at the retention time of 7.5 min, was isolated and examined by TLC/SIMS (Iwabuchi et af., 1987). The prominent ion peaks observed at mlz 427 (positive mode) and 425 (negative mode) indicated a molecular weight of 426, which is coincident with that of the acyl glucuronide of gemfi-
Tables. Peak area ratios for U2/U1 (%) under different storage conditions for two days PH
-20 "C
RTa
7.0 7.5 8.0
1.9 3.6 4.5
67.3 169.7 397.3
a
Room temperature.
brozil. In the negative mode, mlz249 was also observed and assigned as [M - HI- of gemfibrozil. The UV spectroscopic data also supported U1 as being the acyl glucuronide of gemfibrozil. U1 was confirmed as being easily hydrolysed with Glusulasc@to give gemfibrozil. From these results, U1 was established as the 10-acyl glucuronide of gemfibrozil. The lower chromatograms in Fig. 7 show those of the same urine samples after 2 days of standing at room temperature. In addition to peak U1, three furthe peaks were observed (U2, U3 and U4), which werr supposed to be theisomersof U1, i.e., the 2 - 0 - , 3 - 0 or 4-0-acyl glucuronides of gemfibrozil formed by intramolecular rearrangement. The major peak, U2, was also isolated and analysed by UV and TLC/SIMS. These spectra of U2 were quite similar to those of U1. However, U2 could not be hydrolysed with Glusulase@. Since U2 liberated free gemfibrozil by alkaline treatment, U2 was determined as being the positional isomer of U l , the intramolecular rearrangement product. Similarly, U3 and U4 were supposed to be the isomers of U1, because of the disappearance of these peaks and the liberation of gemfibrozil only after alkaline hydrolysis. The peak area ratios of U2 to U1 under different storage conditions are listed in Table 5 . U2 formation was small during thc frozen storage but increased remarkably when kept at room temperature. In both cases, U2 formation depended on the pH of the urine. However, in the present study, as the pH values of urine taken from healthy volunteers were weakly acidic and all samples were stored at -20 "C, the formation of Glusulase@-resistantconjugates was negligible.
~
CONCLUSION Determination of gemfibrozil and its metabolites in plasma and urine by HPLC has been developed and validated. Plasma concentrations of gernfibrozil, M1 and M3 can be determined by fluorescence detection and isocratic elution. In urine, gemfibrozil and its four metabolites can be separated within 20 min using the flow and eluent programming method with two fluorescence detectors in series. The present methods have been used in analytical studies for clinical and biopharmaceutical use. Figure 7. The influences of storage conditions on chromatographic profiles of metabolites i n human urine. Urine samples were adjusted to pH values of 7.0 (left), 7.5 (middle) and 8.0 (right), and stored at -20°C (upper) and room temperature (lower) for 2 days, respectively. Column: YMC-A312 ODS (15 c m x 4 . 6 m m i.d.). eluent: 10 mM acetate buffer (pH 4.711 acetonitrile (64:36), flow rate: 1 m l l m i n .
Acknowledgements We are grateful to Dr. Noriaki Nakaya, Internal Medicinc. Tokyo Hospital, Tokai University, and to Mr. Masayuki Nakamichi, Warner-Lambert K. K. for the clinical studies of gemfibrozil.
SIMULTANEOUS DETERMINATION OF GEMFIBROZIL AND ITS METABOLITES
73
REFERENCES Faed, E. M. (1984). Drug Metabolism Reviews 15, 1213. Forland, S. C., Chaplin, L. and Culter, R. E. (1987). Clin. Chem. 33,
1938. Hengy, H. and Koelle, E. U. (1985). Arzneim.-Forsch./Drug Res.
35, 1637. Iwabuchi, I., Nakagawa, A. and Nakamura, K. (1987).J. Chromatogr. 414, 139. Jain, A. K., Ryan, J. R., Lacorte, W. S. S. and McMahan, F. G. (1981).Clin. Pharmacol. Ther. 29, 254. Nakagawa, A., Nakamura, K. Ishizaki, T. and Chiba, K. (1982). J. Chromatogr. 231, 349.
Nash, D. T. (1980). J. Med. 11, 106. Okerholrn. R. A., Keeley, F. J., Peterson, F. E. and Glazko, A. J. (1976). Proc. R. SOC.Med. 69(Suppl. 2 ) , 1 1 . Randinitis, E. J., Parker Ill, T. D. and Kinkel, A. W. (1984a). J. Chromatogr. 383,444. Randinitis, E. J., Kinkel, A. W., Nelson, C. and Parker 111, T. D. J. Chromatogr. 307,210. (1984b). Tuornilehto, J., Salonen, J. and Kuusisto, P. (1976). Proc. R. SOC. Med. 69(Suppl. 2). 64.
Simultaneous Determination of Gemfibrozil and its Metabolites in Plasma and Urine by a Fully Automated High Performance Liquid Chromatographic System Akihiko Nakagawa", Akemi Shigeta, Haruo Iwabuchi, Masaaki Horiguchi?, Kan-ichi Nakamura and Hidekuni Takahagi Analytical and Metabolic Research Laboratories. Sankyo Co. Ltd., 2-58, Hiromachi 1-chome. Shinagawa-ku, Tokyo 140, Japan
Sensitive and specific methods for the simultaneous determination of gemfibrozil (Lopid@),a lipid-lowering agent, and its metabolites in plasma and urine are described. The methods are based on a fully automated high performance liquid chromatographic (HPLC) system with fluorescence detection. Urine samples, diluted with acetonitrile, were directly analysed by HPLC using a flow and eluent programming method. In the case of plasma, gemfibruzil and its main metabolites were extracted from acidified samples and the resulting extracts injected into the chromatographic system. The sensitivity was approximately 100 ng/mL for gemfibrozil and its four metabolites using 0.5 mL plasma or urine. An acyl glucuronide of gemfibrozil excreted in human urine after oral administration of the drug was isolated and its structure and stability examined.
INTRODUCTION Gemfibrozil, S-(2,S-dimethylphenoxy)-2,2-dimethylpentamic acid (Fig. l), is an oral lipid lowering agent developed by Warner-Lambert (Tuomilehto et al., 1976; Nash, 1980; Jain et al., 1981). The metabolic fate of gemfibrozil was reported by Okerholm et al. (1976) using 3H-labelled compounds. According to this, gemfibrozil is extensively metabolized and excreted in urine as a glucuronide together with four oxidized metabolites (Fig. l), the chief of which is a benzoic acid derivative, M3. For the metabolic profile of gemfibrozil in plasma, nothing has been described except the main
metabolite, M3 (Randinitis et al., 1984a). The determination of gemfibrozil only in plasma and urine can be carried out using gas chromatography (GC) (Randinitis et al., 1984b) and high performance liquid chromatography (HPLC) (Randinitis et a / . , 1984a; Hengy and Koelle, 1985; Forland et al., 1987). The present study describes the simultaneous determination of gcmfihrozil and its metabolites in human plasma and urine by HPLC. Some preliminary results on the metabolic fate of gemfibrozil in humans are also mentioned. The conjugated metabolite of gemfibrozil was elucidated after isolation from urine.
EXPERIMENTAL 73
R1 R2 R3 n __________________________ G e m f ibrozil CH3 H CH3 3 MI CH3 OH CH3 3 M2 CH2OH H CH3 3 M3 COOH H CH3 3 M4 H CHZOH 3 CH3 H CH3 2 c2 CH3 H CH3 5 c5 CH3 Figure 1. Structures of gemfibrozil and its related compounds. * Author to whom correspondence should be addressed iPrcscnt address: Institute of Science and Technology, Inc., 10-2,
Kitashinagawa _?-chome, Shinagawa-ku, Tokyo 140, Japan. 0269-3879/9 1/020M8-06 $05 .oo
01991 by John Wifey s( Sons, Ltd
Chemicals. Gemfibrozil, its four metabolites (Ml-4) and two internal standards (C2, CS) were supplied from Warner-Lambert K. K. (Tokyo, Japan). Glusulase@ was obtained from Dupont (Boston, MA, USA). All solvents and chemicals used were of IIPLC or analytical reagent grade and no further purification was carried out. Apparatus. The IIPLC system used was as follows: Two pumps (LCdA; Shirnadzu, Kyoto, Japan), an automatic sample injector (SIL-hA; Shimadzu), two fluorescence detectors (F-1000; Hitachi, Tokyo, Japan), two integrators (HP3393; Hewlett-Packard, Avondale, PA, USA) and a system controller (SCL-6A; Shimadzu). Peak area data from integrators were processed by an on-line-connected personal computer (PC-9801; NEC, Tokyo, Japan). The flow diagram of the total system employed i s shown in Fig. 2. A fluorescence spectrophotometer (650- 10s: Hitachi). a high-speed muti-wavelength spectrophotomcter (1 IP-1040A; Received 9 April IYW Accepted 14 June I990
SIMULTANEOUS DETERMINATION OF GEMFTBROZIL AND ITS METABOLITES
hY ~
Tablel. Ex and Em wavelengths of maximum fluorescence intensity for gemfibrozil and its four metabolites
-PLOY SIGNAL
Gemfibrozil MI M2
DETECTOR-2
, , INTEGRATOR-2
Figure 2. Flow diagram of a fully automated HPLC system. Thick arrows indicate the sample flow system; broken arrows indicate the operational sequence undergoing the signal.
Hewlett-Packard), and a mass spectrometer equipped with a SLMS ion source (M-80A; Hitachi) were used for qualitative analysis. Chromatographic conditions. For urine samples, the column was a YMC-A312 ODS (15 cm X 4.6 mm I.D.; Yamamura Chemicals, Kyoto, Japan) held at room temperature. The initial mobile phase, consisting of 10 mM acetate buffer (pH4.7)/acetonitrile (55:45), was driven at a flow rate of 1 mL/min. At 10.5 min after sample injection, the mobile phase was changed to a higher acetonitrile content (80%) with a 2 mLImin flow rate. The excitation (Ex) and emission (Em) wavelengths of two detectors were set at 283 and 315 nm, and 300 and 340 nm, respectively, for the simultaneous determination of the drug and its metabolites. A ChH5-1252N(25 cm x 4.6 mm i.d.; Senshu Sci., Tokyo, Japan) column was used for plasma samples at room temperature with 10 mM tartarate buffer (pH 3.3)/acetonitrile/PICBA (4852:O.S) as the eluent at a flow rate of 1mLimin. A fluorescence detector was operated at Ex:293 nm and Em:325 nm. For analysis of the conjugated forms of gernfibrozil, YMC-A312 ODS was used at room temperature with 10 mM acetate buffer (pH 4.7)lacetonitrile (64:36) as the eluent at a flow rate of 1.0 mL/min. Isolation of the conjugates was achieved using the same column with a linear gradient elution (acetonitrile, 0-45%, 30 min). Sample preparation. To assay gemfibrozil and its metabolites in urine excreted as the free form, 0.5 mL acetonitrile containing 10pg/mL C5 as the internal standard was added to 0.5mL urine. The sample was vortexed, centrifuged at 2000 g, and 10 pL of the upper layer was injected into the chromatograph. For the hydrolysis of conjugated gemfibrozil and its metabolites, 0.5 mL Clusulase@solution, diluted with 4 volumes I M acetate buffer (pfl5.2) containing 10% (w/v) sodium metabisulfite (Na2S205),was added to each 0.5mL urine sample. After reciprocal incubation at 37°C for 2 h, 3 mL acetonitrile containing 10 pg/mL C5 was added to the hydrolysate. The mixture was vortexed and centrifuged at 2000 g, and 10 pL of the upper layer was injected into the chromatograph. To the plasma samples (0.5 mL) were added 0.5 mL 1% acetonitrile/phosphate buffered saline (pH 7.4), containing C2 (10 yg/mL) as the internal standard, and 20 pL of formic acid, and the samples were then extracted with 5 mL ethyl acetate/cyclohexane (2:8). After evaporating the solvent, the resulting extracts were reconstructed with 0.5 mL of the eluent and injected to the chromatograph (10 pL).
M3 M4
Ex(nrn)
Ern(nrn)
283 295 280 300 280
315 330 308
340 308
RESULTS AND DISCUSSION Fluorescence spectra For the determination of gemfibrozil and its metabolites by HPLC, fluorescence detection was first introduced in the present study, since these compounds were found to have fluorescence derived from the phenoxy moiety. The maximum excitation (Ex) and emission (Em) wavelengths of these compounds are listed in Table 1. The differences in the wavelength in fluorescence detection in these compounds may be derived from substituents on the phenoxy ring. The detection limit of gemfibrozil was compared between fluorescence and UV detection. UV detection was reconstructed using the same HPLC conditions as described by Hengy and Koelle (1985). At 1ng injection of gemfibrozil to HPLC, the signal-to-noise ratios in the fluorescence and UV peaks were 6.5 and 5.5, respectively. However, the former method was more advantageous in its stability of chromatographic baseline and selectivity for the compounds of interest in body fluids. HPLC separation of the four metabolites of gemfibrozil
The separation of the four metabolites by octadecylsilanized silica (ODS) column was examined using acetate buffers having various pH values and containing 45% acetonitrile as the eluent. The relationships between the pH of the eluent and the capacity factor, k ' , of these metabolites are shown in Fig. 3. The k' values of three monohydroxy isomers of gemfibrozil metabolites,
-A-
M1 M2 & M3
---+-
k'
--
M4
I--
03.5
4.0
5.0
4.5
5.5
6.0
PH
Figure 3. The relationships between pH and k' values of four
metabolites. For HPLC conditions see text.
A . NAKAGAWA
70
'I'ahle 2. The resolutian value (Rs) between M1, M2. M3, and M4 pH
M2-M3
M2-M4
Ml-M4
4.4 4.7 5.0
4.25 8.47 12.83
2.66 2.46 2.12
1.03 1.30 1.70
M I , M2. and M4. were similarly decreased with increasing pHs. The degree of decrease in the k' value of the dicarboxylic acid metabolite, M3, was more steep than the other metabolites. These four metabolites could be separated by the ODS column at pH values from 4.4 to 5.0. At this pH range the resolution values, Rs, are listed in Table 2. The maximum resolution was obtained at pH 5.0. However, the k' value of M3 was so small (0.785) that 1 0 m ~acetate buffer (pH 4.7)iacetonitrile (55:45) was chosen for the elution of these metabolites. Flow programming elution
Since the k' of gemfibrozil was more than 30 under the above HPLC conditions. the eluent was programmed to increase to a higher acetonitrile content ( ~ W O and) a flow rate of 2 mL/min after the elution of these metabolites. This flow programming enabled the elution of both gemfibrozil and the internal standard, C5, within 20min. A drift of the chromatographic baseline of fluorescence detection was not observed before and after the change of eluent and flow rate. Selection of fluorescence wavelength
As shown in Table 1, gemfibrozil and its metabolites give their maximum fluorescence intensities at different wavelengths. Under the above HPLC conditions, Ex at 283 and E m at 315 nm gave the maximum intensity for gemfibrozil, but the peak responses of M1 and M3 were decreased to 80% and lo%, respectively, compared to those obtained by the optimum wavelength for the individual compounds. We solved thi5 problem by utilizing two fluorescence detectors in series. The Ex and Em wavelengths of the first detector were set for determining gemfibrozil, M2, M4, and C5, and the second one, connected in series, was set at 300 (Ex) and 340 (Em) nm for MI and M3, considering the possible effect of diffusion after eluting the column on the separation of the compounds concerned.
Kr A L
Chromatograms of human control and gemfibroziladministered urine after enzymatic hydrolysis are shown in Fig. 4. Small interfering peaks were observed at the retention times of M3 (Fig. 4C), M2 and M4 (Fig. 4A). However, urinary concentrations of these metabolites after thc oral administration of gemfibrozil a t clinical doses were so high that these pcaks did not interfere practically. The amount of liberated gemfibrozil and its four metabolites during enzymatic hydrolysis reached a maximum at 2 h after incubation. After 20 h incubation, the amount of M1 liberated was decreased slightly (ca. 10%) compared to that at 2 h incubation. The hydrolysate with Glusulase@had many interfering peaks when monitored by UV detector. and further purification steps were required for the quantitative analysis. On the other hand, fluorescence detection permitted more selective and sensitive determination than UV, because the fluorescent peaks derived from the enzyme preparation had little effect on the quantitation of gemfibrozil and its metabolites, as shown in Fig. 4. Calibration curves for gemfibrozil and its metabolites were prepared using human control urine spiked with various amounts of standard compounds. Table 3 shows the reproducibility and linearity of the present method, including the enzymatic hydrolysis, for determining the total amounts of these compounds. The values for gemfibrozil and its four metabolites gave satisfactory results for the coefficients of variation (0.999). The quantitative recoveries of these compounds may be due to there being no extraction step in the procedure. The analytical parameters for the free fraction in urine without enzymatic hydrolysis were almost the same as those obtained after enzymatic treatment. Determination of gemfibrozil, M1 and M3 in plasma
The plasma concentrations of the minor metabolites, M2 and M4, wcrc lcss than 30 ng/mL after a 450 mg dose of gemfibrozil in preliminary G U M S quantitation
Determination of gemfibrozil and its metabolites in urine
Gemfibrozil and its metabolites were excreted in urine as conjugates with glucuronic acid, except for M3, which was present mainly as the free form (Okerholm et af., 1976; Randinitis et at., 1984a). Hydrolysis of the conjugates was carried out using Glusulase@at a higher concentration than the conventional method, in the presence of sodium metabisulfite as an antioxidant, to prevent degradation of the compounds (Nakagawa et af., 1982). The hydrolysis rates were checked by the present method.
I I Ir I I . I 1 I 0 5 10 15 20 0 5 10 15 20 Time ( rnin ) Time ( min )
Figure 4. HPLC chromatograms of humar urine samples: (A) and (C)blank urine; (B) and (D) human urine after oral adrninistration of gemfibrozil (450 mglbody). (A)and (B) were detected at Ex:283 nrn and Em:315 nm; (C) and (D) were detected a t Ex:300 nrn and Em:340 nrn.
71
SIMULTANEOUS DETERMINATION OF GEMFIBROLIL AND ITS METABOLITES ~
Table 3. Reproducibility and linearity of the analyses of gemfibrozil and its metabolites in urine . ; Compound
Conc (WgimLl
Coefficient of variation
Conc ranges WmL)
Correlation coefficient
Gemfibrozil MI M2 M3 M4
135 9.7 3.3 22 9 2.9
0.810 1.421 0.668 0.969 0.974
3.00-600 0.64-64 0.15-30 1.80-300 0.15-30
0.999 0.999 0.999 0.999 0.999
"
5 \ Pi 0 1 E
t/2B
1
(hr-1) (l/hr) (hr-I)
0.603
(hr)
1 .41 0.56
Lag time (hr)
10
0.4 7 7 94 1
5
(Nakagawa ef ul., unpublished data), whereas plasma concentrations of gemfbrozil and M3 were estimated to be higher than 100 ng/mL, even 24 h after a single oral administration of the drug at clinical doses (Randinitis et ul., 1Y84a). Since the present HPLC method could not detect concentration levels of less than 100 ng/mL, the simultaneous determination of gcmfibrozil, M1, and M3 was carried out. Several types of chemically modified phases such as C,,, C,, C4, C?, CN, ChH5.and so on. were examined for suitability. The C,H, (pheny1)-bonded phase was found to be the most suitable (Fig. 5 ) . C2 (scc Fig. 1) was chosen as t h e internal standard. Under these IIPLC conditions, M2 anti M4, the two miiior metabolites. would elute between M1 and M3. Figure 5 shows chromatograms of extracts from human plasma (see Experimental Section) after oral administration of gemfihrozil (450 mg/body) when monitored under three different fluorescent conditions. Gemfihrozil, M1 and M3 could be detected with good sensitivity at a wavelength near the maximum fluorescence intensity of M1 (Fig. SA)). In addition. thcse conditions have the advantage that non-separable minor metabolites, M2 and M4, have less effect on the determination of other metabolites. Kecoveries were tested by extracting with 5 mL ethyl acetate/cyclohexanc (2:s) from acidified plasma (0.5 mL) spiked with 5 pg each of gemfibrozil, M1, M3 and C2. These compounds were efficiently extracted. with recovery ratcs of more than 95%. The reproducibility. linearity and sensitivity o f the present method
v 2 4 6
0
Time [ min
-
8
0
1
-
2
4
Time
6
1 min
8
--I 0
2
4
6
8
Time [ win
Figure 5. HPLC chromatograms using CsH, bonded phases. (A), (B) and (C):human plasma after receiving gemfibrozil; (D), ( E l and (F): control human plasma at different fluorescence wavelengths.
0 0
2
4
6
8
12
24
Time
(
hour
)
Figure 6. Plasma concentration-time curves of gemfibrozil, M I and M3 after a single oral administration of the drug (450 mg/ body, n = 6). The pharmacokinetic parameters of gemfibrozil were calculated from mean plasma concentrations.
were also examined using 0.5 mL human plasma spiked with 0.05-25 pg of these compounds. Coefficients of variation expressed as the relative standard deviation (RSD) on the determination of gemtibrozil and M1 were less than 2Y0 in the concentration range of 110 pglmL in plasma. Good correlation coefficients ( r 1 0 . 9 9 9 ) were also obtained for these two compounds in the concentration ranges tested. On the other hand, these parameters for M3 were not as good as those of gemfibrozil and M1. The RSD values for M3 were 4.4 and 4.6% at concentrations of 10 and 1 pglmL, respectively. The correlation coefficient was 0.Y98 in the conccntration range 0.1-50,ugl mL in plasma. However, the present method was still useful for the determination of M3 in plasma.
Practical application for a clinical study
The present methods were applied t o the determination of the plasma concentration and urinary excretion of gemfibrozil and its metabolites after a single oral administration of the drug to six healthy adult malc volunteers (450mg/body). This study was carried out as a part o f a Phase I trial of gemfibrozil. Figure 6 shows the mean plasma concentration-time curves of gemfihro~il,MI and M3. The pharmacokinetic parameters of gemfibrozil were calculated from the mean plasma concentration based on a onecompartment with lag-time absorption model and are listed in Fig. 6. The biological half-life of the drug (1.4 h) was in good agreement with the previously reported data (Randinitis ef af., 1984a). The plasma concentration profile of M3 showed a rather unusual pattern-a plateau at 6-12 h after administration of the drug, with a further increase at 24 h. This seemed attributable to enterohcpatic circulation of this mctabolite (Okerholm el ul., 1976; Randinitis et ul., 1984a). A detailed pharmacokinetic interpretation of M3 in humans will bc mentioned elsewhere. Urinary excretions of gemfibrozil and its metabolites are listcd in Table 4. These values are in good agreement with the data reported by Okerholm er al. (1976) using 3H-labelled gemfbrozil. except for the values obtained for M1 and M-C.
A. NAKAGAWA ETAL
72
Table 4. Determination of 0-24 h urinary gemfibrozil and its metabolites in human (% of dose, 450 mglbody) Compound
Free
Conjugate
Total
M4
0.59 2 0.13 0.08 f 0.01 0.01 f 0.00 15.34 k 2.19 0.01 k 0.00
32.09k 1.30 7.33 k 0.83 0.70 k 0.06 4.51 k0.52 0.50k 0.02
32.68 t 1.35 7.41 f 0.83 0.71 f0.06 19.85+2.18 0.51 20.06
Total
16.04k 2.17
45.13 k 1.57
61.17 k 2.71
Gemfibrozil
M1 M2 M3
Stability of the gemfibrozil conjugate
Gemfibrozil was reported as being excreted in urine as an acyl glucuronide conjugate (Okerholm et al., 1976; Randinitis et al., 1984a, 1984b). The properties of various acyl glucuronides have been reviewed by Faed (1984). According to this, acyl glucuronides are likely to undergo facile intramolecular rearrangement under neutral-to-weak basic conditions, to form 2-0-, 3-0and 4-0-acyl glucuronides. Among them, only 1-0-acyl glucuronide could be the substrate for [email protected] this regard, the properties and stability of the acyl glucuronide of gemfibrozil were examined. Urine samples adjusted to pH values of 7.0, 7.5 and 8.0, respectively, were divided into two portions. One portion was rapidly frozen at -20 "C and the other was kept at room temperature. After two days of storage the urine samples were directly injected into the HPLC system (see Experimental Section). Figure 7 shows chromatograms of these samples under optimal HPLC conditions. The upper chromatograms are those of the urine samples stored at -20°C. The most prominent peak (named as Ul), which eluted at the retention time of 7.5 min, was isolated and examined by TLC/SIMS (Iwabuchi et af., 1987). The prominent ion peaks observed at mlz 427 (positive mode) and 425 (negative mode) indicated a molecular weight of 426, which is coincident with that of the acyl glucuronide of gemfi-
Tables. Peak area ratios for U2/U1 (%) under different storage conditions for two days PH
-20 "C
RTa
7.0 7.5 8.0
1.9 3.6 4.5
67.3 169.7 397.3
a
Room temperature.
brozil. In the negative mode, mlz249 was also observed and assigned as [M - HI- of gemfibrozil. The UV spectroscopic data also supported U1 as being the acyl glucuronide of gemfibrozil. U1 was confirmed as being easily hydrolysed with Glusulasc@to give gemfibrozil. From these results, U1 was established as the 10-acyl glucuronide of gemfibrozil. The lower chromatograms in Fig. 7 show those of the same urine samples after 2 days of standing at room temperature. In addition to peak U1, three furthe peaks were observed (U2, U3 and U4), which werr supposed to be theisomersof U1, i.e., the 2 - 0 - , 3 - 0 or 4-0-acyl glucuronides of gemfibrozil formed by intramolecular rearrangement. The major peak, U2, was also isolated and analysed by UV and TLC/SIMS. These spectra of U2 were quite similar to those of U1. However, U2 could not be hydrolysed with Glusulase@. Since U2 liberated free gemfibrozil by alkaline treatment, U2 was determined as being the positional isomer of U l , the intramolecular rearrangement product. Similarly, U3 and U4 were supposed to be the isomers of U1, because of the disappearance of these peaks and the liberation of gemfibrozil only after alkaline hydrolysis. The peak area ratios of U2 to U1 under different storage conditions are listed in Table 5 . U2 formation was small during thc frozen storage but increased remarkably when kept at room temperature. In both cases, U2 formation depended on the pH of the urine. However, in the present study, as the pH values of urine taken from healthy volunteers were weakly acidic and all samples were stored at -20 "C, the formation of Glusulase@-resistantconjugates was negligible.
~
CONCLUSION Determination of gemfibrozil and its metabolites in plasma and urine by HPLC has been developed and validated. Plasma concentrations of gernfibrozil, M1 and M3 can be determined by fluorescence detection and isocratic elution. In urine, gemfibrozil and its four metabolites can be separated within 20 min using the flow and eluent programming method with two fluorescence detectors in series. The present methods have been used in analytical studies for clinical and biopharmaceutical use. Figure 7. The influences of storage conditions on chromatographic profiles of metabolites i n human urine. Urine samples were adjusted to pH values of 7.0 (left), 7.5 (middle) and 8.0 (right), and stored at -20°C (upper) and room temperature (lower) for 2 days, respectively. Column: YMC-A312 ODS (15 c m x 4 . 6 m m i.d.). eluent: 10 mM acetate buffer (pH 4.711 acetonitrile (64:36), flow rate: 1 m l l m i n .
Acknowledgements We are grateful to Dr. Noriaki Nakaya, Internal Medicinc. Tokyo Hospital, Tokai University, and to Mr. Masayuki Nakamichi, Warner-Lambert K. K. for the clinical studies of gemfibrozil.
SIMULTANEOUS DETERMINATION OF GEMFIBROZIL AND ITS METABOLITES
73
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35, 1637. Iwabuchi, I., Nakagawa, A. and Nakamura, K. (1987).J. Chromatogr. 414, 139. Jain, A. K., Ryan, J. R., Lacorte, W. S. S. and McMahan, F. G. (1981).Clin. Pharmacol. Ther. 29, 254. Nakagawa, A., Nakamura, K. Ishizaki, T. and Chiba, K. (1982). J. Chromatogr. 231, 349.
Nash, D. T. (1980). J. Med. 11, 106. Okerholrn. R. A., Keeley, F. J., Peterson, F. E. and Glazko, A. J. (1976). Proc. R. SOC.Med. 69(Suppl. 2 ) , 1 1 . Randinitis, E. J., Parker Ill, T. D. and Kinkel, A. W. (1984a). J. Chromatogr. 383,444. Randinitis, E. J., Kinkel, A. W., Nelson, C. and Parker 111, T. D. J. Chromatogr. 307,210. (1984b). Tuornilehto, J., Salonen, J. and Kuusisto, P. (1976). Proc. R. SOC. Med. 69(Suppl. 2). 64.
