Simultaneous Determination of Testosterone Metabolites in Liver Microsomes Using Column-Switching Semi-microcolumn High-Performance Liquid Chromatography

Analytical Biochemistry 295, 248 –256 (2001) doi:10.1006/abio.2001.5223, available online at http://www.idealibrary.com on

Simultaneous Determination of Testosterone Metabolites in Liver Microsomes Using Column-Switching Semimicrocolumn High-Performance Liquid Chromatography Shuko Tachibana 1 and Makoto Tanaka Drug Metabolism and Physicochemical Property Research Laboratory, Daiichi Pharmaceutical Company Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan

Received March 28, 2001; published online July 19, 2001

A sensitive and selective column-switching semimicrocolumn high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of testosterone and eight of its metabolites (6␣-, 6␤-, 16␣-, 16␤-, 7␣-, 2␣-, and 2␤-hydroxytestosterone, and androstenedione) in liver microsomes. After incubation for 10 min, testosterone and its metabolites were extracted from the microsomes with ethyl acetate, and the extract was evaporated to dryness. The residue was dissolved in the mobile phase and loaded onto the HPLC system. The analytes were first concentrated in a precolumn and subsequently transferred to the analytical column, where they were separated using linear gradient elution. A UV detector set at 254 nm was used to detect the analytes. This newly developed method clearly separated TES and the metabolites with high resolution and was found to be reproducible with intra- and interday variability of <10.7%. This method has been subsequently used to determine the testosterone hydroxylation activities catalyzed by 15 different recombinant CYP isozymes. The results confirmed the formation of stereoselectively hydroxylated metabolites by each CYP isozyme. © 2001 Academic Press Key Words: HPLC; column switching; testosterone; cytochrome P450; human liver microsomes; cytochrome b 5.

Cytochrome P450 (CYP) 2 enzymes play an important role in the oxidation of both xenobiotic compounds, 1 To whom correspondence should be addressed. Fax: 81-3-56968332. E-mail: [email protected]. 2 Abbreviations used: CYP, cytochrome P450; HPLC, high-performance liquid chromatography; b 5 , cytochrome b 5 ; TES, testosterone;

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such as drugs, and endogenous substrates, such as steroids (1–3). Testosterone (TES) is one such endogenous substrate for CYPs, and its regio- and stereospecific hydroxylation has been used as an assay for specific CYP isozyme activities. In particular, 6␤-hydroxylation of TES is frequently used to determine CYP3A isozyme activity (4 –9). Therefore, a robust, sensitive, and selective method for the determination of TES metabolites is essential for this assay to be useful. Many high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) methods have been reported for the separation of TES and its hydroxylated metabolites (4 –16). However, some of these HPLC methods cannot produce baseline separation of all the metabolites, while TLC methods require radioactively labeled TES. In recent years, microcolumn liquid chromatography techniques have been developed; often this technique is used together with mass spectrometry or other analytical techniques (17). Microcolumn and semi-microcolumn HPLC have many advantages over conventional HPLC such as higher sensitivity and reduced mobilephase consumption. On the other hand, low sample loadability (in volume) and extra-column band broadening had previously limited the widespread application of these techniques. However, recent reports indicate that column-switching techniques that use a low dead-volume valve unit can surmount these problems (18 –21). Consequently, column-switching techniques

TLC, thin-layer chromatography; LC, liquid chromatography; AD, androstenedione; G-6-P, glucose 6-phosphate; G-6-P DH, glucose-6phosphate dehydrogenase; ␤-NADPH, nicotinamide adenine dinucleotide phosphate (reduced); THF, tetrahydrofuran; IS, internal standard; UV, ultraviolet; QC, quality control; CV, coefficient of variation; RE, relative error. 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

HPLC DETERMINATION OF HYDROXYTESTOSTERONES IN MICROSOMES

have become very useful for on-line sample clean-up and enrichment of analytes, and have been widely adopted for the analysis of drugs, such as unstable antibiotics in biological samples (22–24). This paper describes the development of a new HPLC method for the determination of TES and eight TES metabolites, 2␣-, 2␤-, 6␣-, 6␤-, 7␣-, 16␣-, and 16␤hydroxytestosterone, and androstenedione (AD), in rat and human liver microsomes. By using a columnswitching semi-microcolumn system, baseline separation of TES and its metabolites was achieved with increased selectivity and sensitivity, which enabled the detection of even minor metabolites. This newly developed method was then used to measure the TES hydroxylation activities of human liver microsomes and recombinant CYP isozymes. MATERIALS AND METHODS

Chemicals TES and ethyl acetate were purchased from Nakalai Tesque, Inc. (Kyoto, Japan). 6␣-, 6␤-, 16␣-, 16␤-, 7␣-, 2␣-, and 2␤-Hydroxytestosterone were obtained from ULTRAFINE Chemicals (Manchester, England). 4-Androstene-3,17-dione and 11-deoxycortisol were purchased from Sigma Chemical Co. (St. Louis, MO). Glucose 6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-P DH), and ␤-NADPH were purchased from Oriental Yeast, Co., Ltd. (Tokyo, Japan). Ketoconazole and cimetidine were purchased from Research Biochemicals International (Natick, MA). HPLC-grade acetonitrile, methanol, and tetrahydrofuran (THF) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). All other chemicals were analytical reagent grade and used without further purification. Deionized water purified with a Milli-Q system (Waters Association, Millipore, Milford, MA) was used. Microsomes Human liver microsomes (H0610, pool of 15 donors) were obtained from XenoTech L.L.C. (Kansas City, KS). Instead of human liver microsomes, rat liver microsomes were used in the validation study for ethical reasons. Rat liver microsomes were prepared by differential centrifugation as previously described (25). A Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., CA) was used to determine protein concentrations with bovine serum albumin as the standard (26). Microsomes prepared from insect cells transfected with a baculovirus expressing one of the following human CYPs [CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9*2(Cys 144), 2C18, 2C19, 2D6, 2E1⫹b 5, 3A4, 3A4⫹b 5, 3A5, and 4A11] and human NADPH-cytochrome P450 reductase (Supersomes) were obtained from Gentest Corp. (Worburn, MA). Recombinant rat

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CYP2A1 in microsomes from B-lymphoblastoid cells coexpressing NADPH-cytochrome P450 reductase was also purchased from Gentest Corp. The CYP contents were used as described in the data sheets provided by the manufacturers. Preparation of Standard Solutions Stock solutions of TES and its metabolites were prepared in methanol and stored at ⫺20°C. The working standard solutions were prepared by further diluting the stock solutions with methanol. Incubation of TES with Microsomes The incubation mixture for microsomal TES hydroxylation activity assay (total volume 0.5 ml) contained 50 mM potassium phosphate buffer (pH 7.4), liver microsomes (0.25 mg protein) or recombinant CYP microsomes (50 pmol CYP), TES (3.13–1000 ␮M), magnesium chloride (5 mM), G-6-P (12 mM), G-6-P DH (1.1 units), and ␤-NADPH (1 mM). The reaction was started by adding ␤-NADPH and stopped by adding 5 ml of ice-cold ethyl acetate after incubation for 10 min at 37°C. The internal standard (IS), 11-deoxycortisol (50 ␮l of 0.025 mg/ml in methanol solution) was then added to each sample. After 5 minutes of vigorous shaking followed by centrifugation (1500g, 10 min), the organic phase was isolated and evaporated to dryness by centrifugal evaporation. The residue was dissolved in 100 ␮l of solvent A, consisting of acidified Milli-Q water (adjusted to pH 3.5 with acetic acid):methanol (70:30, v/v), and then filtered through an Ultrafree membrane filter (C3GV, 0.22 ␮m, Millipore), before being loaded onto the HPLC system (50 ␮l). In the case of standard samples used to establish the calibration curves, TES (final concentrations: 0.1, 0.5, 1, 5, 10, 25, and 50 ␮M) and its metabolites (final concentrations: 0.1, 0.25, 0.5, 1, 5, 10, and 25 ␮M) were spiked with incubation mixtures before addition of liver microsomes. Next, ethyl acetate (the reaction quencher), liver microsomes, and the IS solution were added in this order, and the standard samples were extracted as described previously. Instruments and Chromatographic Conditions A schematic diagram of the column-switching semimicrocolumn HPLC system is shown in Fig. 1. The HPLC system consisted of an LC-10AD pump (pump 1; Shimadzu Corp., Tokyo, Japan) and instruments from the NANOSPACE SI-1 series (Shiseido Co., Ltd., Tokyo, Japan), which consisted of two 2001 gradient pumps (pump 2), a 2010 degasser, a 2002 variablewavelength ultraviolet (UV) detector, a 2003 autosampler, and a 2011 high-pressure six-port switching valve. The UV detector was set at 254 nm. The au-

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tosampler and the six-port switching valve were used to provide automated on-line sample enrichment and analysis. A CAPCELLPAK C18 UG120 column (35 ⫻ 1.5 mm i.d., 5 ␮m particle size, Shiseido Co., Ltd.) was used as the precolumn. The mobile phase for the precolumn was Milli-Q water (pH 3.5). A TSK-gel ODS-80Ts column (150 ⫻ 2.0 mm i.d., 5 ␮m particle size, Tosoh Corp., Tokyo, Japan) was used as the analytical column. A linear gradient of solvent A [Milli-Q water (pH 3.5):methanol (70:30, v/v)] and solvent B [Milli-Q water (pH 3.5):methanol:THF (10:85:5, v/v/v)] was used as the eluent for the analytical column. The total flow rate through the column was set at 0.2 ml/min. An ultrasonic bath was used to degas solvents A and B before use. The column temperature was ambient. Column-Switching Procedure Details of the column-switching procedure are shown in Table 1. Step 1 (0 –5 min). The six-port switching valve was initially set at position A. A microsomal extract was loaded onto the precolumn. The precolumn was eluted with Milli-Q water (pH 3.5) at a flow rate of 0.2 ml/min (pump 1). At the same time, the analytical column was being equilibrated with a 70:30 (v/v) mixture of solvent A and B at a flow rate of 0.2 ml/min (pump 2). Step 2 (5–35 min). The valve was switched to position B and the analytical mobile phase was pumped through the precolumn in the backflush mode. The components retained on the precolumn were eluted onto the analytical column, where the analytes were separated at a flow rate of 0.2 ml/min for 30 min. The elution was performed using a linear gradient mixture of solvents A and B starting with a 70:30 (v/v) mixture and ending with a 30:70 (v/v) mixture. Step 3 (35– 45 min). The valve was again switched to position A to equilibrate the precolumn and analytical column for 10 min before injection of the next sample. Calibration Curves The calibration curves for TES and its metabolites were obtained by least-squares linear regression of the peak area ratios of the standard to the IS versus the concentrations of each standard. Recovery The absolute recovery of the analytes and the IS from the incubation mixtures was determined by comparing the peak areas of standard solutions to those of rat microsomal extracts spiked with standard solutions at the same concentrations.

Selectivity Microsomal extracts that had not been spiked with any analyte were prepared from five different rats and from pooled human microsomes (pool of 15 donors). These extracts were assayed according to the previously described procedures to evaluate the selectivity of the method. Precision and Accuracy Intraday precision and accuracy of the method were determined by replicate analyses (n ⫽ 6) of the quality control (QC) samples (0.1, 0.5, 5, and 25 ␮M for metabolites and 0.1, 1, 10, and 50 ␮M for TES). Interday precision and accuracy were determined by assaying the QC samples on 5 different days. Precision was evaluated from the calculated coefficient of variation (CV). Accuracy was evaluated from the relative error (RE), determined by comparing the measured concentration to the theoretical concentration. Kinetic Analysis Kinetic study for TES 6␤-hydroxylation by human liver microsomes was carried out at TES concentrations ranging from 3.13 to 1000 ␮M. Michaelis-Menten kinetic parameters were estimated using a computer program KaleidaGraph version 3.08 (Synergy Software, Reading, PA). The inhibitory constants (K i) of ketoconazole and cimetidine on the TES 6␤-hydroxylation activity in human liver microsomes were determined using the Dixon plot ([I] vs 1/[V]) (27). RESULTS

Construction of a Column-Switching System To achieve highly selective separation of TES and its many hydroxylated metabolites, a column-switching semi-microcolumn HPLC system was constructed as shown in Fig. 1. Ethyl acetate extracts of samples (50 ␮l) were initially chromatographed onto the precolumn, which concentrated the analytes. By rotating the six-port switching valve, the analytes retained on the precolumn were directly transferred to the analytical column in the back-flush mode. These analytes were then separated by linear gradient elution. Well-defined chromatographic peaks for TES and each of its metabolites studied were obtained as shown in Fig. 2. Ethyl acetate extraction and the columnswitching system provided adequate clean-up of microsomal samples; this is clearly shown in Fig. 2A by the absence of interfering endogeneous peaks. Neither microsomal extracts from five individual rats nor pooled human microsomes had any interference with the assay, indicating the high selectivity of the method. A typical chromatogram of extracts from rat microsomes

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HPLC DETERMINATION OF HYDROXYTESTOSTERONES IN MICROSOMES

FIG. 1. Schematic diagram of the column-switching HPLC system. Analytes are first concentrated in the precolumn during step 1 and then transferred to and separated by the analytical column during step 2.

spiked with TES and its metabolites is shown in Fig. 2B. The retention times of 6␣-, 7␣-, 6␤-, 16␣-, 16␤-, 2␣-, and 2␤-hydoxytestosterone, AD, TES, and IS were 12.8, 14.7, 15.4, 16.7, 18.7, 20.4, 21.3, 24.4, 27.9, and 22.6 min, respectively. Consequently, this method achieves baseline separation of all the compouds. Linear gradient elution was used for the separation, but no significant change occurred in the baseline. These results illustrate that the present analytical method has satisfactory resolution of TES and its metabolites, which is necessary for the simultaneous determination of these compounds. Validation of Analytical Method The absolute recoveries of TES and its metabolites from rat liver microsomes were 71.1–107.6% at concentrations of 0.5, 5, and 25 ␮M (1, 10, and 25 ␮M for TES). At the minimum concentration (0.1 ␮M), the recoveries of these compounds ranged widely from 60.5 to 113.8%. The recovery of the IS was 82.0% ⫾ 1.2% (n ⫽ 15). Thus, ethyl acetate extraction and a columnswitching method can be used to extract these compounds from liver microsomal incubates.

Calibration curves for TES and its metabolites obtained on 5 different days were linear over the concentration range of 0.1–25 ␮M for metabolites and 0.1–50 ␮M for TES (Table 2). The correlation coefficients (r 2 ) ranged from 0.9824 ⫾ 0.0044 to 0.9993 ⫾ 0.0004. The interday CV of the slopes of the calibration curves was ⬍7.1%. Good correlation and slope reproducibility were observed for each compound. The results for intraday precision and accuracy of the method are shown in Table 3. The CV ranged from 0.7 to 5.0% and the RE ranged from 86.7 to 110.5% for QC concentrations higher than 0.5 ␮M. The lower limit of quantitation of all the compounds using 0.5 ml of microsomal incubates was determined to be the concentration of the lowest calibration standard (0.1 ␮M), which exhibited a CV of ⬍9.7%, and a RE ranged from 96.9 to 117.6%. The results for interday precision and accuracy are shown in Table 4. The CV was ⬍10.7% and the RE ranged from 89.8 to 108.3%. The results indicate that this method is highly sensitive and has acceptable inter- and intraday precision and accuracy.

TABLE 1

Column-Switching Procedure 2 Gradient pumps (2)

Step 1 Step 2 Step 3 (wash)

Time (min)

Valve position

Pump flow rate (ml/min)

0–5 5–35 35–45

A B A

0.2 0.2 0.2

Pump (1) H 2O (pH 3.5) (%)

Solvent A (%)

Solvent B (%)

100 100 100

70 70 to 30 70

30 30 to 70 30

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TACHIBANA AND TANAKA

concentrations (from 0.1 to 1.0 mg/ml). Michaelis-Menten kinetic analysis for TES 6␤-hydroxylation in human liver microsomes revealed a K m value of 68.6 ␮M and a V max value of 4.8 nmol/min/mg protein (11.4 nmol/min/nmol CYP). These values are similar to values reported previously (28, 29). Figure 2C shows a chromatogram of human liver microsomal extracts after incubation with TES (100 ␮M). 6␤-Hydroxytestosterone was identified as the major metabolite produced. The other minor metabolites seen were 2␤-hydroxytestosterone, AD, and an unidentified peak at 13.1 min. This unidentified peak is thought to be 15␤-hydroxytestosterone, which was not identified because we could not obtain an authentic standard (6). This method has also been used to evaluate the inhibitory effects of drugs and new chemical entities on CYP3A activity. It is well known that ketoconazole and cimetidine are potent inhibitors of CYP3A4 in vivo (30 –34). Therefore, an in vitro inhibition study was performed, and this method was used to measure TES 6␤-hydroxylation activity in the presence of either ketoconazole or cimetidine. The results show that the TES 6␤-hydroxylation catalyzed by CYP3A4 was inhibited by both ketoconazole (K i ⫽ 0.06 ␮M) and cimetidine (K i ⫽ 417 ␮M). TES Hydroxylation by Recombinant Human and Rat CYP Isozymes

FIG. 2. Representative HPLC chromatograms obtained from (A) human liver microsomes; (B) extracts from rat microsomes spiked with all hydroxytestosterones (10 ␮M), TES (25 ␮M), and IS (11deoxycortisol); and (C) human liver microsomes after incubation with TES (100 ␮M) for 10 min in the presence of ␤-NADPH. The chromatograms are shown at a full scale of 500 mV.

Kinetics of TES 6␤-Hydroxylation by Human Liver Microsomes TES 6␤-hydroxylation is known to be selectively catalyzed by the CYP3A family, and has been frequently used as a probe to measure CYP3A activity in vitro (6 –9). The present method was used to determine whether it would be useful in detecting TES 6␤-hydroxylation activity in human liver microsomes (pool of 15 donors) over a TES concentration range of 3.13– 1000 ␮M. Preliminary experiments were performed to verify that TES 6␤-hydroxylation activitiy was proportional to the incubation time (0 –30 min) and the protein

Next, this highly selective method was used to determine the TES hydroxylation activities catalyzed by 14 recombinant human CYP isozymes expressed in baculovirus-insect cell systems and one rat recombinant CYP isozyme expressed in a B-lymphoblastoid cell line, all of which coexpress NADPH-cytochrome P450 reductase. Incubations were carried out for 30 min at a substrate concentration of 50 ␮M. The results (Table 5) show that human CYP3A4 is the main CYP that is responsible for catalyzing TES 6␤- and 2␤-hydroxylation. CYP3A5, 1A1, and 2D6 also can catalyze 6␤-hydroxylation, but not to the same extent as CYP3A4. The 6␤-hydroxylation catalyzed by CYP3A4 increases 3-fold in the presence of b 5 compared to the activity in the absence of b 5. This confirms previous observations that b 5 enhances the TES 6␤hydroxylation activity catalyzed by CYP3A4 (35–38). Interestingly, however, the 2␤-hydroxylation activity catalyzed by CYP3A4 remained the same whether b 5 was present or absent. To our knowledge, this is the first observation that 2␤-hydroxylation catalyzed by CYP3A4 was not affected by the presence of b 5 . Regarding the other TES metabolites, recombinant CYP2C19 catalyzes the formation of AD to a greater extent than any other CYP isozyme tested; this result is consistent with the result previously reported by

253

HPLC DETERMINATION OF HYDROXYTESTOSTERONES IN MICROSOMES TABLE 2

Interday Variation of Calibration Curves (Weighting Factor: 1/conc 2) Correlation coefficient (r 2 )

Slope Calibration concentration range (␮M)

Mean ⫾ SD (n ⫽ 5)

CV (%)

Mean ⫾ SD (n ⫽ 5)

CV (%)

0.1–25 0.1–25 0.1–25 0.1–25 0.1–25 0.1–25 0.1–25 0.1–25 0.1–50

15.06 ⫾ 0.4 18.88 ⫾ 0.6 14.42 ⫾ 0.2 12.50 ⫾ 0.4 15.07 ⫾ 0.8 16.09 ⫾ 0.3 13.35 ⫾ 1.0 15.08 ⫾ 0.8 15.04 ⫾ 0.4

2.7 3.0 1.6 3.3 5.5 2.1 7.1 5.5 2.4

0.9893 ⫾ 0.0162 0.9937 ⫾ 0.0032 0.9944 ⫾ 0.0020 0.9824 ⫾ 0.0044 0.9951 ⫾ 0.0063 0.9993 ⫾ 0.0004 0.9888 ⫾ 0.0150 0.9956 ⫾ 0.0038 0.9843 ⫾ 0.0055

1.6 0.3 0.2 0.4 0.6 0.0 1.5 0.4 0.6

6␣-Hydroxytestosterone 6␤-Hydroxytestosterone 16␣-Hydroxytestosterone 16␤-Hydroxytestosterone 2␣-Hydroxytestosterone 2␤-Hydroxytestosterone 7␣-Hydroxytestosterone Androstenedione Testosterone

Yamazaki and Shimada (39). Human CYP2B6 moderately catalyzed 16␤-hydroxylation, but CYP2B6 might contribute only slightly to TES hydroxylation in human liver microsomes because it is not present in human liver in large amounts (7, 40 – 42). Finally, 6␣-, 2␣-, and 7␣-hydroxylation reactions were not catalyzed by any human CYP, but the results showed that rat CYP2A1 catalyzed 7␣-hydroxylation and, to a much lesser extent, 6␣-hydroxylation of TES. DISCUSSION

The objective of this study was to develop a method that can clearly separate TES metabolites. Although a semi-microcolumn HPLC method with direct injection to the analytical column was tried initially, a columnswitching system was adopted to achieve more selective separation of the analytes. This technique is also useful for excluding or separating analytes from interfering peaks. In particular, the method provides a means of avoiding interference from CYP3A inhibitors

during inhibition studies. Sample enrichment and concentration of the analytes on the precolumn by columnswitching greatly improved the resolution of the method, and resulted in baseline separation of all TES metabolites. As compared with a direct injection method, the faster eluting compounds, especially 6␣and 6␤-hydroxytestosterones eluted in sharper peaks. Furthermore, the column-switching method prevented the rapid deterioration of the analytical column, and consequently, the resolution was maintained for over 400 samples. The separation of 6␤- and 7␣-hydroxytestosterone was the major test for the selection of an analytical column and optimization of the mobile phase for the desired separation. All TES metabolites separated on the TSK analytical column with high resolution, but the separation was not completely achieved with other columns tested. Additionally, a mobile phase composed only of Milli-Q water and MeOH was used initially, but THF was added to solvent B to reduce the elution time

TABLE 3

Intraday Precision and Accuracy (n ⫽ 6) QC 0.1 ␮M

6␣-Hydroxytestosterone 6␤-Hydroxytestosterone 16␣-Hydroxytestosterone 16␤-Hydroxytestosterone 2␣-Hydroxytestosterone 2␤-Hydroxytestosterone 7␣-Hydroxytestosterone Androstenedione Testosterone a b c

QC 0.5 ␮M

QC 5 ␮M

QC 25 ␮M

CV (%)

RE (%)

CV (%)

RE (%)

CV (%)

RE (%)

CV (%)

RE (%)

7.8 5.1 5.1 3.1 8.1 6.9 1.1 9.7 1.9

96.9 100.2 96.8 101.4 104.4 102.8 117.6 103.2 111.4

1.7 1.4 1.4 2.1 4.0 2.0 2.2 5.0 4.9 a

101.3 102.9 107.4 106.6 104.9 105.2 110.4 103.6 97.4 a

2.2 0.7 1.7 1.7 1.2 1.1 1.3 0.9 1.1 b

95.1 100.4 96.7 93.5 96.1 110.5 92.0 99.1 100.9 b

1.0 1.5 0.9 2.2 0.8 0.8 1.0 2.3 1.2 c

90.0 92.2 93.2 89.2 96.5 91.8 93.0 95.2 86.7 c

Added concentration at 1 ␮M. Added concentration at 10 ␮M. Added concentration at 50 ␮M.

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TACHIBANA AND TANAKA TABLE 4

Interday Precision and Accuracy (n ⫽ 5) QC 0.1 ␮M

b c

QC 5 ␮M

QC 25 ␮M

CV (%)

RE (%)

CV (%)

RE (%)

CV (%)

RE (%)

CV (%)

RE (%)

6.4 5.3 5.0 9.1 3.4 1.4 5.1 3.6 10.7

95.7 96.9 95.4 92.8 98.4 101.6 93.3 98.1 95.0

4.4 5.3 4.0 3.5 6.3 5.3 6.2 6.7 4.7 a

105.0 98.8 105.9 105.9 102.1 99.1 108.3 102.8 102.4 a

2.6 4.9 4.5 2.1 3.9 3.4 4.5 4.2 3.2 b

95.1 102.2 98.6 94.7 97.0 106.9 92.8 99.3 101.9 b

6.1 3.2 2.2 4.4 3.2 1.3 8.2 3.6 3.3 c

94.3 93.9 94.2 89.8 95.8 99.8 96.8 95.4 89.9 c

6␣-Hydroxytestosterone 6␤-Hydroxytestosterone 16␣-Hydroxytestosterone 16␤-Hydroxytestosterone 2␣-Hydroxytestosterone 2␤-Hydroxytestosterone 7␣-Hydroxytestosterone Androstenedione Testosterone a

QC 0.5 ␮M

Added concentration at 1 ␮M. Added concentration at 10 ␮M. Added concentration at 50 ␮M.

and to increase peak resolution. With 5% THF in the mobile phase (the only THF concentration tested), a run time was shortened by approximately 6 min, peak heights of later eluting compounds increased, and peak separation improved, compared to results for the mobile phase without THF. The limits of quantitation for all the compounds examined in this study were 0.1 ␮M. A scale-up of the total volume of incubation mixture and the HPLC injection volume might increase the sensitivity of this column-switching method without loss of resolution. Several HPLC methods have been reported for the separation of TES and its metabolites (4 – 8, 10 –16). Most of these methods have some disadvantages such as insufficient resolution of all the metabolites, peak broadening of later eluting compounds, and baseline changes caused by complex gradient systems. The present method has overcome these problems by application of the column-switching technique to semi-microcolumn HPLC, although the time required for the chromatographic separation is 30 min.

The results of this study show that this method can be used for the determination of the TES hydroxylation activities in liver microsomes, and also for screening potential CYP3A inhibitors. Additionally, the TES hydroxylation results obtained for microsomes containing a recombinant CYP isoform confirmed the previously reported stereoselectivity for each CYP isozyme. Moreover, our results showed that 2␤-hydroxylation catalyzed by CYP3A4 was not enhanced in the presence of b 5 compared to the absence of b 5 . Several studies have shown that CYP3A4 catalytic activities for TES 6␤hydroxylation and nifedipine oxidation were stimulated by the addition of b 5 to microsomes expressing a recombinant CYP3A4. It is thought that electron transfer from NADPH-cytochrome P450 reductase to b 5 is an important step in certain CYP3A4 catalyzed oxidations (35–38). It would be interesting to know whether the effect of b 5 on CYP activity is regioselective, stereoselective, or both. In conclusion, a sensitive and selective analytical method for the determination of TES and its metabo-

TABLE 5

Testosterone Hydroxylation by Recombinant Human and Rat CYP Enzymes a Formation of testosterone metabolites (nmol/min/nmol CYP)

6␣-Hydroxylation 7␣-Hydroxylation 6␤-Hydroxylation 16␣-Hydroxylation 16␤-Hydroxylation 2␣-Hydroxylation 2␤-Hydroxylation 17-Oxidation (AD) a

1A1

1A2

2A6

2B6

2C8

2C9

2C18

2C19

2D6

2E1

3A4

3A4⫹b 5

3A5

4A11

rat 2A1

— — 1.16 — — — — 0.07

— — 0.06 — — — — 0.10

— — — — — — — —

— — — — 0.24 — — 0.04

— — — 0.10 — — — —

— — — — 0.05 — — 0.04

— — — — — — — 0.06

— — — — 0.11 — 0.04 1.03

— — 0.16 — — — — 0.47

— — — — — — — —

— — 2.22 — — — 0.32 —

— — 6.92 0.16 — — 0.31 —

— — 0.60 — — — 0.07 —

— — — — — — — 0.08

0.08 2.11 — — — — — —

Testosterone (50 ␮M) was incubated with recombinant CYP (50 pmol) for 30 min in total volume 0.5 ml.

HPLC DETERMINATION OF HYDROXYTESTOSTERONES IN MICROSOMES

lites in microsomes was established by combining semi-microcolumn HPLC and column-switching systems. The present method permits the separation of TES and its metabolites with good resolution and high sensitivity, and is capable of measuring even minor metabolites. This method has proved to be suitable for the evaluation of TES hydroxylation by CYP isozymes in humans and rats, and is also applicable to a CYP3A inhibition study.

13.

14.

15.

ACKNOWLEDGMENT The authors thank Mr. Steve E. Johnson for editing this manuscript.

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