Determination of Testosterone Metabolites in Rat Hepatocytes with and without Cryopreservation by On-Line SPE Column-Switching LC and MS Detection

Determination of Testosterone Metabolitesin Rat Hepatocytes with and withoutCryopreservation by On-Line SPEColumn-Switching LC and MS Detection

Gerhard Friedrich1,&, Thorsten Rose1, Alexander Wawkuschewski1, Sabine Kafert-Kasting2,Britta Laube2, Lubomir Arseniev2, Klaus Rissler3

1 Pharmbiodyn, Institute of Contract Research, P. O. Box 1108, 79207 Denzlingen, Germany; E-Mail: [email protected] Cytonet GmbH & Co. KG Hannover Branch, Feodor Lynen Strasse 21, 30625 Hannover, Germany3 Brunnlirain 5, 4125 Riehen, Switzerland

Received: 21 May 2007 / Revised: 8 November 2007 / Accepted: 8 November 2007Online publication: 12 December 2007

Abstract

In a recently published paper development of a sensitive automated ‘‘on-line’’ solid-phase extraction (SPE)/RP-HPLC assay for 6b-hy-droxytestosterone (6b-OHT) with corticosterone as the internal standard (IS) was reported and its potential for quantification of varioustestosterone metabolites in culture media reflecting metabolic activity of cultured human and animal hepatocytes demonstrated [1]. In thisfollowing contribution the technique has been extended to determination of another five testosterone metabolites in cultured rat hepatocytesusing an identical ‘‘on-line’’ SPE/RP-HPLC procedure and detection by tandem MS-MS with an atmospheric pressure chemical ionization(APCI) source in the selected reaction monitoring (SRM) mode as that described in [1]. All six testosterone metabolites, namely 2a-OHT,2b-OHT, 6a-OHT, 6b-OHT, 7a-OHT and 16a-OHT, could be sufficiently separated from each other and thus an unequivocal assignment tothe individual structures was achieved. Validation data are presented specifying the limits of quantitation as well as the mean values of thecoefficients of variation (CV) for the target analytes and the accuracy obtained at five different days. Regio- and stereoselective testosteronehydroxylation by rat hepatocytes was measured in a long-term culture system with and without exposure to rifampicin as an inducer of liverCYP 3A4 activity. In addition, testosterone hydroxylation was analyzed in cultures of cryopreserved hepatocytes that had been stored at�196 �C. The rat hepatocytes were cultured after thawing for up to 11 days and induction of testosterone hydroxylase activity could bedemonstrated in cultures which underwent a new cryopreservation protocol.

Keywords

Column liquid chromatographyOn-line SPE/LC-MSTestosterone metabolitesHydroxylase activitiesHepatocytes

Introduction

Cytochrome P 450 (CYP) isoenzymes,

which all are members of the haemo-

protein family, play an essential role in

biotransformation of either drugs or

endogenous substrates [2–21]. Testoster-

one is one of the most important

endogenous targets for the action of liver

enzymes, undergoing various pathways

of biotransformation, which in the case

of cytochrome oxidase P 450 3A4 (CYP

3A4) preponderantly yields 6b-hydroxy-This paper is dedicated to Professor HinrichCramer on the occasion of his 75th birthday.

2008, 67, 31–39

DOI: 10.1365/s10337-007-0485-20009-5893/08/01 � 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

Original Chromatographia 2008, 67, January (No. 1/2) 31

testosterone (6b-OHT) as the main

metabolite [13, 14, 16, 18]. In the same

way 6b-OHT also represents the main

testosterone metabolite in hepatic micro-

somes of untreated rats accounting for

about two thirds of total metabolites [14].

‘‘In vitro’’ studies using mammalian

hepatocytes are generally considered as

a valuable tool for prediction of in vivo

metabolism with special focus on the

prediction of xenobiotic biotransforma-

tion patterns in the liver. When relying

on cell culture systems for the analysis

of unknown biotransformation path-

ways, e.g., of a drug candidate molecule,

it is important to assure the metabolic

competence of the hepatocytes in cul-

ture by measuring reference metabolic

activities. In this respect, testosterone

metabolism is frequently used to assess

CYP 3A4 activity by analysis of

6b-OHT generation. CYP 3A4, which

alone comprises an average of 70% of

the total intestinal CYP content [2], is

of special interest since a majority of

xenobiotics is converted by this enzyme

complex.

Another important feature concern-

ing the quality of in vitro hepatocyte sys-

tems is the inducibility of enzyme

activities. CYP isoenzymes are inducedby

a variety of compoundswidely differing in

structural features. In this respect, phe-

nobarbital [12], 3-methylcholanthrene

and Aroclor 1254 [13], the latter com-

posed of a mixture of chlorinated biphe-

nyls, as well as rifampicin [14] are widely

chosen as the standard inducers of

CYP 3A4 activity. However, important

species differences are known to exist

as studied by Kern et al. who also found

a substantial effect of rifampicin on

6b-testosterone hydroxylase activity in

cultured human hepatocytes, whereas

no effect was reported in rat hepato-

cytes [14].

Besides 6b-OHT, a number of posi-

tional isomers are generated from tes-

tosterone by action of CYP isoenzymes

different form CYP 3A4. In this respect,

CYP 2C11 was shown to produce

7a-OHT [23, 24], 16b-OHT [23, 24],

16a-OHT [17, 23] and 2a-OHT [17,

21–24] the latter activity also generated

by CYP 3A2 [17], CYP 1A1 and CYP

3A1 [22, 23]. Although CYP 3A4 still

represents the main pathway of 6b-OHT

formation, a wide variety of other CYP

isoenzymes, such as, e.g., CYP 1A1, [22],

CYP 1A2 [22], CYP 3A1 [9, 14, 17,

21–24], CYP 3A2 [9, 17, 24], CYP 2A2

[17], CYP 2C13 [17] as well as CYP 3A3

and CYP 3A5 [18] contribute to

6b-hydroxylation. CYP 1A1 [22], CYP

2A1 [17, 18, 22–24], CYP 2A2 [17] and

CYP 2B1/2 [23, 24] are responsible for

oxidization of testosterone to 7a-OHT

and, in addition, CYP 2A2 also yields

12b-OHT [18], 15a-OHT [17] and

18-OHT [17]. Besides 6b-OHT, 2b-OHT

and 15b-OHT were generated by action

of CYP 3A1 [17, 21, 23, 24] and CYP

3A2 [23, 24], whereas CYP 2A4/5

participates in 15b-hydroxylation [24]. In

addition to the action of CYP 2C11

[17, 22–24], hydroxylation of testoster-

one to 16a-OHT is also catalyzed by

CYP 2B1 [17, 18, 21, 23, 24] and CYP

2B2 [21, 23, 24]. Interestingly CYP

2B1 [21, 23] and CYP 2B2 [21, 23, 24]

also participate in 16b-OHT formation

[17, 18, 21, 23].

A compilation of hitherto used

chromatographic procedures for either

identification or quantitative determi-

nation of a variety of testosterone

metabolites is given in [1] and the

reader is referred to this paper to get

more comprehensive information. In the

same paper development of a highly

specific ‘‘on-line’’ SPE/column-switch-

ing gradient reversed-phase HPLC as-

say for determination of 6b-OHT in

media mimicking the situation in cul-

tured human hepatocytes is reported.

In this contribution the method is

extended to quantitative determination

of another five testosterone metabolites

from cultured rat hepatocytes. As

already successfully applied previously,

both exact structural assignment and

quantitative determination was done by

tandem LC-MS-MS in the selected

reaction monitoring (SRM) mode. In

this respect 2a-, 2b-, 6a-, 6b-, 7a- and

16a-OHT using corticosterone as the

internal standard (IS), were detected and

quantified. The method was applied to

quantitation of these testosterone

metabolites in rat hepatocytes. The

hepatocyte cultures were characterized

over time, with and without rifampicin

induction, and with and without cryo-

preservation.

Experimental

Reagents and Materials

The testosterone metabolites 2a-OHT,

2b-OHT, 6a-OHT, 6b-OHT, 16a-OHT

and corticosterone as the IS were ob-

tained from Sigma-Aldrich (Steinheim,

Germany), whereas 7a-OHT was re-

ceived from Ultrafine Chemicals

(Manchester, UK). Methanol and

2-propanol (both gradient grade) and

formic acid (analytical grade) were

purchased from Merck (Darmstadt,

Germany) and triethylamine (puriss,

p. a.) from Fluka (Buchs, Switzerland).

DMSO for medical purpose was from

WAK Chemie (Steinbach, Germany).

For cell culturing experiments fetal calf

serum from Biochrom (Berlin,

Germany) and HepatoZym from Gibco

(Eggenstein, Germany) were used. Dul-

becco’s modified eagle medium (DMEM

High Glucose) was obtained from

Cell Concepts (Umkirch, Germany).

The mixture of b-glucuronidase and

arylsulfatase was from Roche Diagnos-

tics (Mannheim, Germany) and rifam-

picin from Sigma-Aldrich (Steinheim,

Germany). High purity water for the use

in HPLC was prepared with a Milli-Q

reagent water systemTM from Millipore-

Waters (Milford, MA, USA). A BioTrap

500 MSTMSPE-column (20 · 4 mmI.D.)

from Chromtech (Hagersten, Sweden)

and a Prontosil 60-5 C18-H (250 · 2 mm

I. D., 5 lm particle size) analytical

column from Bischoff (Leonberg,

Germany), were used for sample extrac-

tion and separation, respectively.

Preparation of Hepatocytesand Sample Generation fromCell Culture Supernatants

Hepatocytes were prepared and cultured

at Cytonet GmbH & Co. KG, branch

Hannover. The rat hepatocytes were

isolated from male Wistar WKY rats as

described by Hengstler et al. [25]. Cryo-

preservation was performed following

the protocol published earlier [26]. For

induction experiments, the cells were

cultured in the presence of 50 lMrifampicin for 24 h prior to the testos-

32 Chromatographia 2008, 67, January (No. 1/2) Original

terone hydroxylation assay performed at

day 3 and 8, respectively. Prior to addi-

tion of testosterone, the cultures were

washed once with serum free medium

and subsequently incubated for one hour

in the presence of 250 lM testosterone.

After 60 min aliquots of 800 lL of the

cell culture supernatant were removed

and hydrolysed with 100 Fishman units

of b-glucuronidase and 800 Roy units of

arylsulfatase at pH 4.5 for 2 h at 37 �Cwith agitation. The samples were then

shock-frozen in liquid nitrogen and

stored at �80 �C.The concentration C (ng mL�1) of

the respective monohydroxy-testosterone

(OHT) species was determined byHPLC-

MS-MS as described below and the

hydroxylation activities (pMol min�1)

were calculated as follows: activity =

C · V · f/t · M, taking into account

the molecular mass (M = g mol�1) of

OHT, the incubation time (t = 60 min),

the volume of the cell culture superna-

tant (V = 1 mL) and an experimental

factor resulting from supernatant dilu-

tion during hydrolyzation (f = 1.1335).

The calculation of testosterone hydrox-

ylation activity refers to all viable cells in

the well from which the supernatant was

derived.

Sample Preparation,Chromatographic Separationand Detection

For preparation of calibration samples,

IS and quality controls as well as

extraction of the target analytes from the

biological matrices using a column-

switching device, the same protocol as

that described previously [1] was applied.

In brief, a solution of the IS corticoste-

rone (1 mg mL�1) was dissolved in

methanol/water 1:1 (v,v) and diluted to

20 lg mL�1 with DMEM High Glucose.

The different testosterone metabolites

were prepared in a corresponding way to

yield final dilutions of 30, 50, 100, 250,

500, 1000, 2000, 4000 ng mL�1 for the

calibratorsand250, 500and1000 ng mL�1

for the quality controls, except for

7a-OHT, where the ranges for calibra-

tion and controls were 5, 15, 30, 50, 100,

250, 500, 1000 ng mL�1 and 5, 15, 30, 50

and 100 ng mL�1, respectively. Prior to

sample preparation 50 lL of IS

(20 lg mL�1) was added to 500 lL of

calibration solutions, quality controls

and supernatants of cultured rat hepa-

tocytes from which 100 lL were

subjected to the ‘‘on-line’’ SPE/column-

switching RP-HPLC procedure. Analyte

enrichment by means of a BioTrap

500 MSTM SPE column was carried

out with 10 mM triethylammonium

formate buffer (pH 6). Back-flush of

analyte onto the analytical C18 column

and subsequent chromatographic sepa-

ration was accomplished in the gradi-

ent mode with mobile phase A composed

of water–methanol–tetrahydrofuran

(73:20:7, v/v/v) and mobile phase B

composed of methanol and THF

(93:7, v/v) at a flow-rate of 0.2 mL

min�1. Signal monitoring was effected by

tandem MS-MS detection in the SRM

mode. The individual experimental con-

ditions of the column-switching protocol

comprising pre-column sample extrac-

tion, back-flush onto the analytical col-

umn, final chromatographic separation

0 5 10 15 20 25 30 35

Time (min)

0

1

2

3

4

5

6

7

8

9

10

0

1

2

3

4

5

6

7

8

9

10

7

2

1

6

4

3

5

Fig. 1. HPLC-MS (SRM) chromatograms of the monohydroxy testosterone metabolites1 = 2a-OHT, 2 = 2b-OHT, 3 = 6a-OHT, 4 = 6b-OHT, 5 = 7a-OHT, 6 = 16a-OHT onthe upper trace and and 7 = the internal standard corticosterone on the lower trace


in the gradient mode and mass spectro-

metric detection are completely identical

to those reported previously [1].

Results

Sample Extraction,Chromatographic Separationand Detection

Using the chromatographic procedure

described previously [1], all members of

a standard mixture of the six analytes to

be determined as well as the IS cortico-

sterone were sufficiently separated from

each other (Fig. 1) and assignment of the

HPLC signals could be unambiguously

effected by means of their characteristic

retention times under the applied exper-

imental conditions. However, it should

be mentioned that the pairs 7a-/15a-OHT, 2b-/11b-OHT and 6b-/19-OHT

cannot be separated from each other

using the applied chromatographic

system. Nevertheless, as shown in our

research group (unpublished observa-

tions), at least partial separation of all

these critical pairs can be accomplished

when switching to a more shallow gradi-

ent profile and a substantial increase

in run time. However, induction of

15b-OHT, 11b-OHT and 19-OHT does

not play a significant role after CYPP 450

enzyme induction of hepatocytes ob-

tained from the investigated species

[20–22]. As a consequence, chromatog-

raphy described in [1] effecting satisfac-

tory separation of the six target hydroxy

Table 1. Precision and accuracy data of 2a-OHT (a), 2b-OHT (b), 6a-OHT (c), 6b-OHT (d), 7a-OHT (e) and 16a-OHT (f)

270602 010902 021002 241002 021202Calc. amount(ng mL�1)

Calc. amount(ng mL�1)




Mean SD CV (%) Acc. (%)

a: Validation data of 2a-OHT250 161.9 254.5 250.9 235.9 244.8 241.9 33.6 13.9 96.8250 293.5 257.6 248.8 250.0 221.2 254.2 25.9 10.2 101.7500 442.6 316.8 446.8 475.4 512.2 458.3 67.7 14.8 91.7500 n. d. 460.2 429.8 472.7 568.0 482.7 59.7 12.4 96.51000 970.2 1020.6 1172.5 816.4 979.1 1047.8 120.6 11.5 104.81000 1056.4 1093.3 1191.5 1199.0 979.1 1103.9 93.1 8.4 110.4

b: Validation data of 2b-OHT250 188.0 239.4 252.5 226.5 253.6 238.3 20.8 8.7 95.3250 248.8 251.3 247.9 251.9 223.2 244.6 12.1 4.9 97.9500 307.0 320.4 436.2 492.1 544.6 451.2 87.2 19.3 90.2500 432.2 462.1 457.6 470.9 589.1 482.4 61.4 12.7 96.51000 1043.0 955.3 1137.3 844.3 1016.5 1037.1 99.8 9.6 103.71000 1046.4 1102.0 1107.4 1171.3 947.7 1075.0 83.8 7.8 107.5

c: Validation data of 6a-OHT250 286.6 223.8 284.7 319.6 273.1 271.7 37.5 13.8 108.7250 243.4 224.8 276.1 336.0 249.3 265.9 43.3 16.3 106.4500 462.2 539.5 522.5 576.0 472.1 490.4 63.6 13.0 98.1500 416.7 393.6 460.5 583.4 477.2 466.3 73.5 15.8 93.31000 956.2 950.7 1189.9 1170.7 991.2 1060.0 100.7 9.5 106.01000 1044.6 948.5 1027.6 1181.0 1140.0 1068.3 92.8 8.7 106.8

d: Validation data of 6b-OHT250 219.7 230.6 256.2 264.9 282.0 250.3 21.1 8.4 100.1250 279.7 233.0 254.4 248.1 234.5 250.0 18.9 7.6 100.0500 404.4 551.5 482.2 523.5 520.3 491.7 52.1 10.6 98.3500 492.5 413.8 463.5 555.7 509.6 487.0 52.8 10.8 97.41000 1152.8 979.2 1090.3 960.1 1111.0 1056.1 63.1 6.0 105.61000 1104.9 1005.1 1019.4 1052.5 1085.3 1053.4 42.3 4.0 105.3

e: Validation data of 7a-OHT5 5.2 5.0 5.3 5.2 4.6 5.0 0.3 5.3 101.015 13.9 – 12.2 12.1 16.7 13.7 2.1 15.6 91.530 26.5 35.6 35.5 32.9 30.5 32.2 3.8 11.9 107.350 58.0 44.4 56.6 56.0 46.6 52.3 6.3 12.0 104.6100 95.2 93.8 97.2 118.1 95.6 100.0 10.2 10.2 100.0

f: Validation data of 16a-OHT250 222.7 227.0 241.7 284.1 274.3 253.4 22.1 8.7 101.3250 261.7 266.4 226.4 267.7 261.5 256.7 17.2 6.7 102.7500 395.3 487.8 440.1 524.8 537.1 485.2 53.1 10.9 97.0500 469.1 437.0 466.1 537.5 557.7 493.5 51.5 10.4 98.71000 1024.1 1005.8 987.2 955.6 1105.0 1023.9 48.4 4.7 102.41000 1056.2 1028.9 964.8 1026.9 1084.1 1032.2 44.3 4.3 103.2

ND not determined


testosterone metabolites was not changed

and sensitive quantitative determination

of the six testosterone metabolites was

achieved by mass spectroscopy in the

SRM mode exploiting the transitions

m/z 305.3 (267.7–270.7) for all testos-

terone monohydroxy metabolites and

m/z 347.1 (327.7–330.7) for the IS

corticosterone [1].

Linearity, Specificity, Precisionand Accuracy, Limitof Detection

An exhaustive validation protocol for

6b-OHT has been published in [1] and

therefore, only a short one is given here.

As in the case of 6b-OHT [1], linearity

was achieved in the concentration range

of 100–4000 ng mL�1 for 2a-OHT,

2b-OHT, 6a-OHT, 6b-OHT and

16a-OHT using a total of eight calibra-

tors as well as 5–1000 ng mL�1 for of

7a-OHT (for individual concentrations

see ‘‘Experimental’’). In all cases the

mean values for the correlation factor r2

were >0.99 (mean = 0.9985). As de-

picted in Table 1a–f, mean values of the

coefficients of variation (% CV) for the

target analyte obtained for the concen-

trations 250, 500 and 1000 ng mL�1 at

five different days over a period of

5 months ranged from 8.4–14.8% for

2a-OHT, 4.9–19.3% for 2b-OHT,

8.7–16.3% for 6a-OHT, 4.0–10.8% for

6b-OHT and 4.3–10.9% for 16a-OHT.

The corresponding values for the accu-

racy were 91.7–110.4% for 2a-OHT,

90.2–107.5% for 2b-OHT, 93.3–108.7%

for 6a-OHT, 97.4–105.6% for 6b-OHT

and 97.0–103.2% for 16a-OHT. In the

case of 7a-OHT, where substantially

lower concentrations are encountered

compared with the other metabolites un-

der investigation, control measurements

have been performed at the 5, 15, 30, 50

and 100 ng mL�1 level. The mean values

for either%CV and accuracy as shown in

Table 1e ranged from 5.3–15.6% and

91.5–107.3%, respectively. If concentra-

tions of metabolites are expected to be

very low, higher sample volumes are to be

subjected to the SPE procedure. In this

way using 1000 lL of sample the limits of

quantitation can be decreased to 1.0, 0.7,

0.4, 2.0, 0.5 and 1.5 ng, respectively for

2a-OHT, 2b-OHT, 6a-OHT, 6b-OHT,

7a-OHT and 16a-OHT.

Testosterone HydroxyMetabolites Determinedin Supernatants from RatHepatocyte Cultures

One representative chromatogram show-

ing the metabolic profile attributable to

rat hepatocytes is depicted in Fig. 2.

As shown in Fig. 3, memory effects

during long term chromatography are

avoided using the tandem approach

technique, i. e., column A and column B

in change, which proved to be the case in

thousands of runs.

Quantitative analysis was performed

with samples generated at culture days 0,

1, 3 and 8 for rat hepatocytes with and

without cryopreservation. Please note

that culture day 0 means the day of cell

isolation for the cell cultures not under-

going cryopreservation and the day of

thawing for the cryopreserved cultures.

For the cell preparation shown, at day 0

the cryopreserved cultures displayed

elevated enzyme activities as compared

to the freshly prepared hepatocytes (see

Fig. 3). This phenomenon was observed

0 5 10 15 20 25 30 35 40

Time (min)

0

1

2

3

4

5

6

7

8

9

10

0

1

2

3

4

5

6

7

8

9

10

8

3

5

4 6

9

1

2

7

Fig. 2. Representative chromatogram of testosterone metabolites obtained from rat hepatocytesafter induction with rifampicin; peaks 8 and 9 are attributable to hitherto unknownmonohydroxy testosterone metabolites; peak numbering 1–7 as in Fig. 1


in about 50% of the cultures analyzed.

Meanwhile, activities of cultures with

and without cryopreservation were

comparable at day 1 and day 3 and

depression of enzyme activity in the

cryopreserved cultures was observed at

day 8 for 6b-OHT, 16a-OHT, 2a-OHT

and 2b-OHT. While the maximum cul-

ture time after thawing was 11 days for

one specific cell preparation, data ob-

tained 8 days after thawing were reliably

achieved (data not shown).

In addition to the basal levels of en-

zyme activity, the inducibility of a given

enzyme activity is generally considered as

reflection of well conserved function of in

vitro cell culture systems [27]. Therefore,

at culture day 3 and 8 the rat hepatocyte

cultures were also analyzed after 24 h of

incubation in the presence of the inductor

rifampicin (see Fig. 4 and data not

shown). In the cultures without cryo-

preservation, activities generating

all OHT metabolites except 6a-OHT

and 16a-OHT were induced (induc-

tion � twofold). Interestingly, induction

was observed at day 8 only and was

most pronounced for 6b-OHT and

2b-OHT where fourfold induction was

observed. Notably, induction was also

observed using cultures after cryopreser-

vation and the induction factors achieved

were in the same range (up to fourfold) as

in the fresh cultures although on a lower

level of absolute activity values.

Discussion

As reported in the section ‘‘Results’’, the

fully automated ‘‘on-line’’ SPE-tandem

column-switching RP-HPLC method

with tandem MS-MS detection using an

APCI source in the SRM mode previ-

ously developed for 6b-OHT [1] was

successfully transferred to the quantita-

tive determination of 2a-, 2b-, 6a-, 6b-,7a- and 16a-OHT in cultured hepato-

cytes of mammalian species. The proce-

dure described in more detail in [1] was

the first technique which guarantees that

no ghost-peaks appeared in the chro-

matogram, which means that memory

effects can be completely avoided, as

demonstrated in Fig. 3. It is effected by

cleaning the second SPE column for

sample enrichment as well as the second

analytical column while analytical sepa-

ration currently runs on the first one. At

the rentention time of the IS the back-

ground of both hydroxy-testosterone

detection and blank is slightly elevated.

This effect is present in all chromato-

grams. Nevertheless it does not interfere

with quantification of the hydroxy-tes-

tosterone metabolites. The reason for

this effect is attributable to an unspecific

fragmentation of the IS, which is only

visible due to the high concentration of

the IS. That means that the slight ele-

vation of the background is not caused

by a memory effect.

The BioTrap 500 MSTM SPE-column

used for sample enrichment and cleaning

pertains to the class of so-called

0

0

2

4

6

8

10

0

2

4

6

8

10

0

2

4

6

8

10

0

2

4

6

8

10

NL: 1,00E6

m/z= 268,6-269,6 F:

+ p APCI SRM ms2

305,30@29,00 [

267,70-270,70] MS

CYTO 05 12 146

NL: 1,00E7

m/z= 328,7-329,7 F:

+ p APCI SRM ms2

347,20@29,00 [

327,70-330,70] MS

CYTO 05 12 146

NL: 1,00E6

m/z= 268,6-269,6 F:

+ p APCI SRM ms2

305,30@29,00 [

267,70-270,70] MS

cyto 05 12 148

NL: 1,00E7

m/z= 328,7-329,7 F:

+ p APCI SRM ms2

347,20@29,00 [

327,70-330,70] MS

cyto 05 12 148

Column ARun 146

Column ARun 148

142 6 3810 18

Time (min)

34302622

Fig. 3. A blank chromatogram (column A, run 148) compared to a sample chromatogram(testosterone metabolites after incubation with human hepatocytes as described in the section‘‘Results’’, column A, run 146) is shown. In both cases the scale range is 1.00 · 106. The internalstandards are in the scale range 1.00 · 107


‘‘restricted access materials’’ (RAM-

phases) permitting access to the inner

pore structure only for low molecular

weight molecules while high molecular

weight ones, such as proteins are diverted

to waste [28, 29]. By this measure clog-

ging is extensively avoided and long-term

application guaranteed before replace-

ment of these materials and therefore the

method is excellently suited for metabolic

investigations. In all those studies using

the novel tandem ‘‘on-line’’ SPE tech-

nique it could be shown that indeed no

memory effects occurred. To our

knowledge no tandem SPE/analytical

separation column system as that one

reported in this paper has been published

hitherto. In an analoguous commercially

available system memory effects are only

prevented by the use of a unique SPE

column per sample and use of only a

single separation column. Therefore

possible memory effects are not pre-

vented in the separation column. In our

work however, sample pretreatment is

performed using only a single SPE

column for a large number of samples.

Cleaning of this SPE column as well of

the corresponding separation column is

done using the tandem technique (two

SPE and two separation columns).

Therefore, it is obvious that the benefit

drawn from the novel ‘‘on-line’’ tandem

SPE procedure lies in the fact that both

pre-treatment of a second sample and

column equilibration is done during

separation of the first one which saves

time and money. In this respect it should

be emphasized that numerous analyses

16α Testosterone hydroxylase activity of rat hepatocytes

0

50

100

150

200

250

300

n im/lle

w/lo

mp

n im/lle

w/lo

mp

6β Testosterone hydroxylase activity of rat hepatocytes

0

50

100

150

200

250

300

D 0

Days in culture

a b


0

50

100

150

200

250

300

2β Testosterone hydroxylase activity of rat hepatocytes

0.00

50.00

100.00

150.00

200.00

250.00

300.00

D 0

Days in culture

nim/lle

w/lo

mp

nim/lle

w/lo

mp

c d


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.67α Testosterone hydroxylase activity of rat hepatocytes

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

nim/lle

w/lo

mp

fe

D 1 D 3 D 8 D 8 ind

D 0

Days in culture

D 1 D 3 D 8 D 8 ind D 0

Days in culture

D 1 D 3 D 8 D 8 ind

D 0

Days in culture

D 1 D 3 D 8 D 8 ind

without cryopreservationafter cryopreservation




D 8 indD 8D 3D 1

D 0

Days in culture

D 1 D 3 D 8 D 8 ind



n im/lle

w/lo

mp

Fig. 4. Testosterone hydroxylation activities generating 2a-OHT (a), 2b-OHT (b), 6a-OHT (c), 6b-OHT (d), 7a-OHT (e) and 16a-OHT (f),respectively, as determined from the supernatant of rat hepatocyte cultures with and without cryopreservation and, for samples from day 8, withand without rifampicin induction (induction, dotted colums). Cell seeding was adjusted such that nearly confluent cultures resulted in 6-well-platesin both fresh and cryopreserved cultures. However, the activities are not normalized to cell numbers. Note that the scale differs between 4a–d and4e–f


can be done on the SPE columns before

they have to be replaced whereas in

contrast, every sample needs a new SPE

cartridge applying the aforementioned

automated technique, which results in

additional analysis costs. Another

advantage of the new ‘‘on-line’’ SPE

procedure is given by a high degree of

reproducibility because a lot of pipetting

steps each contributing to individual

errors are replaced by the fully auto-

mated procedure. Last but not least

handling of problematic samples, i. e.,

those being susceptible to oxidative or

light-induced decomposition as well as

light-induced isomerism is markedly re-

duced because sample pretreatment takes

place in the dark in a closed system.

With respect to application of this

procedure to measurement of biological

samples, quantitation of the six testos-

terone metabolites allows for either

investigation of different culture condi-

tions or donor characterization and can

also be used as an early quality control

assessment, e.g., by measuring the

induction of enzyme activities at day 3.

The method was used for culture super-

natant from rat and human hepatocytes.

The data obtained from human hepato-

cyte preparations have been reported

and discussed earlier [29] and therefore

are not further mentioned here. The

measurements from rat hepatocyte cul-

ture supernatant were equally successful

in specific detection and quantification

of 2a-, 2b-, 6a-, 6b-, 7a- and 16a-OHT

indicating basal levels of the respective

testosterone hydroxylation activities.

Moreover, incubation of the rat hepa-

tocytes in the presence of rifampicin led

to induction of the OHT activities, most

pronounced for 6b-OHT and 2b-OHT

(see Fig. 4).

We have used the sandwich model

[30] as the basis of our cryopreservation

protocol and obtained rat and human

hepatocyte cultures that were viable and

metabolically active at least until day 3

after thawing. All testosterone metabo-

lism activities found in the fresh cultures

were reflected in the cryopreserved

counterparts, including induction (see

Fig. 4). While the absolute activities

were well conserved in rat cultures, the

level was generally reduced in human

hepatocytes [30]. Since the inducibility of

CYP activities is generally considered as

reflection of well conserved function of

in vitro cell culture systems [31], it is of

note that the induction factors achieved

after cryopreservation were in the same

range (up to fourfold) as in the fresh

cultures.

In both rat and human cultures, in-

creased formation of 2a-, 2b-, 6b-, 7a-and 16a-OHT after rifampicin treat-

ment was observed while 6a-OHT gen-

erating activities obviously were not

induced. Other authors have reported

more complex observations, e.g., a

marked increase in cytochrome P 450

oxidase activity occuring after treatment

of rats with, e.g., phenobarbital, 3-

methylcholanthrene and Aroclor 1254,

while 6b-OHT formation decreases but

nevertheless it still remains the principal

testosterone metabolite [14]. A similar

influence of phenobarbital, 3-methyl-

cholanthrene and other kinds of

inducers, such as pregnenolone-16-car-

bonitrile (PCN), dexamethasone, eryth-

romycin estolate, troleandromycin and

spironolactone was reported by

Sonderfan et al. [32]. Due to interac-

tions within the CYP 450 family, inte-

gration of induction and inhibition

phenomena can occur resulting in

complex metabolic patterns. Therefore,

analysis of the six testosterone hydroxy

metabolites as reported in this study

can be used as an indicator system

when hepatocytes are incubated with

known and unknown substances. In this

respect quantification of testosterone

hydroxy metabolites by automated ‘‘on-

line’’ SPE/RP-HPLC can be very useful

for the attribution of induction profiles

to well characterized inducers such as

rifampicin, but also to so far unchar-

acterized substances, e.g., in preclinical

drug development. In addition, the

technology is also suitable for analysis

of hepatocyte-specific metabolic com-

petence when tracking stem cell differ-

entiation towards the hepatocyte

phenotype.

Conclusions

Analysis of the six testosterone hydroxy

metabolites as reported in this study can

be used as an indicator system when

hepatocytes are incubated with known

and unknown substances using quanti-

fication of the resulting testosterone hy-

droxy metabolites by automated ‘‘on-

line’’ SPE/RP-HPLC. The previously

published ‘‘on-line’’ tandem SPE/LC-

MS-MS method for quantitative deter-

mination of 6b-hydroxytestosterone(6b-OHT) [1] could be successfully

extended to measurement of another

five testosterone metabolites, namely

2a-OHT, 2b-OHT, 6a-OHT, 7a-OHT

and 16a-OHT in common with 6b-OHT.

All six metabolites could be well sepa-

rated from each other and quantified

with high sensitivity being sufficient for

quantitation of them in biological fluids,

i. e., in supernatants of cultured human

and rat hepatocytes. The fully auto-

mated sample preparation procedure

tremendously reduces both carryover

effects and possible sample degradation

during processing. In addition, due to

minimization of pipetting steps repro-

ducibility of analytical information is

markedy enhanced allowing processing

of large sample numbers with a mini-

mum of man power. Last but not least it

could be shown that cryopreservation is

a suitable means to preserve hepatocytes

for biopharmaceutical and metabolic

studies and that induction proves to be

an important indicator for preserved

functionality of hepatocytes also after

cryopreservation.

References

1. Friedrich G, Rose T, Rissler K (2003)J Chromotogr B 784:49

2. Engman HE, Lennerhas H, TaipalensuuJ, Otter C, Leidvik B, Artursson P (2001)J Pharm Sci 90:1736

3. Thomas PE, Reik LM, Ryan DE, LevinW (1981) J Biol Chem 256:1044

4. Ryan DE, Thomas PE, Reik LM, LevinW (1982) Xenobiotica 12:727

5. Guengerich FP, Martin MV, Beaune PH,Kremers P, Wolff T, Waxman DJ (1986)J Biol Chem 261:5051

6. Waxman DJ, Attisano C, Guengerich FP,Lapenson DP (1988) Arch Biochem Bio-phys 263:424

7. Guengerich FP, Shimada T (1991) ChemRes Toxicol 4:391

8. Nelson DR, Koymanns L, Kamataki T,Stegemann JJ, Feyereisen R, Waxman DJ,Waterman MR, Gotoh O, Coon MJ, Es-tabrook RW, Gunsalus IC, Nebert DW(1996) Pharmacogenetics 6:1


9. Waxman DJ, Ko A, Walsh C (1983) J BiolChem 258:11937

10. Levin W, Thomas PE, Ryan DE, WoodAW (1987) Arch Biochem Biophys258:630

11. Waxman DJ (1988) Biochem Pharmacol37:71

12. van der Hoven T (1981) Biochem BiophysRes Comm 100:1285

13. Wood AW, Ryan DE, Thomas PE, LevinW (1983) J Biol Chem 258:8839

14. Kern A, Bader A, Pichlmayr R, SewingK-F (1997) Biochem Pharmacol 54:761

15. Gebhard R, Hengstler JG, Muller D,Glockner R, Buenning P, Laube B, Sch-melzer E, Ullrich M, Utesch D, Hewitt N,Ringel M, Reder-Hilz B, Bader A, Lang-sch A, Koose T, Burger HJ, Maas J,Oesch F (2003) Drug Metab Rev 35:145

16. Pelkonen O, Maenpaa J, Taavitsainen P,Rautio A, Raunio H (1988) Xenobiotica28:1203

17. Arlotto MP, Trant JM, Estabrook RW(1991) Methods Enzymol 206:454

18. Smith SJ, Korzewka KR, Aoyama T,Gonzalez FJ, Darbyshire JF, SugiyamaK, Gillette JR (1992) Drug Metabol Dis-pos 20:566

19. Woertelboer HM, de Kruif CA, van IerselAAJ, Falke HE, Noordhoek J, BlaauboerBJ (1990) Biochem Pharmacol 40:2525

20. Hengstler JG, Ringel M, Biefang K,Hammel S, Milbert U, Gerl M, KlebachM, Diener B, Platt KL, Bottger T, Stein-berg P, Oesch F (2000) Chem Biol Interact125:51

21. Ringel M, Oesch F, Gerl M, Klebach M,Quint M, Bader A, Bottger T, HengstlerJG (2002) Xenobiotica 21:653

22. Chung WG, Roh HK, Kim HM, Cha YN(1998) Chem Biol Interact 113:1

23. Bullock P, Pearce R, Draper A, Povdal J,Bracken W, Veltman J, Thomas P, Par-kinson A (1995) Drug Metabol Dispos23:736

24. Parkinson A, Clement RP, Casciano CN,Cayen MN (1992) Biochem Pharmacol43:2169

25. Hengstler JG, Utesch D, Steinberg P,Platt KL, Diener B, Ringel M, Swales N,Fischer T, Biefang K, Gerl M, Bottger T,Oesch F (2000) Drug Metab Rev 32:81

26. Reese JA, Byard JL (1981) In Vitro 17:93527. Li AP, Gorycki PD, Hengstler JG, Ked-

deris GL, Koebe HG, Rahmani R, deSousas G, Silva JM, Skett P (1999) ChemBiol Interact 121:117

28. Cassian NM, Lima VV, Oliveira RV, dePietro AC, Cass QB (2006) Anal BioanalChem 384:1462

29. Souverain S, Rudaz S, Veuthey J-L (2004)J Chromatogr B 801:141

30. Kafert-Kasting S, Alexandrova K, Bart-hold M, Laube B, Friedrich G, ArseniewL, Hengstler JG (2006) Toxicology220:117

31. Dunn JCY, Yarmush ML, Koebe HG,Tompkins RG (1989) FASEB J 3:174

32. Sonderfan AJ, Arlotto MP, Dutton D,McMillen SK, Parkinson A (1987) ArchBiochem Biophys 255:27


Documents

Determination of Testosterone Metabolites in Rat Hepatocytes with and without Cryopreservation by On-Line SPE Column-Switching LC and MS Detection