CHIRALITY 4:509-514 (1992)

Direct Coupled Column Separation and Determination of the Diastereomeric Glucuronides of Almokalant, a New Class 111 Antiarrhythmic Drug, in Human Urine MORGAN STEFANSSON AND KURT-mGEN HOFFMANN Department of Analyticd Pharmaceutical Chemistly, Uppsala University, Biomedical Center, Uppsala, Sweden (MS.) and Astra Hiissle Research Laboratories, Department of Phamzacokznetics and Drug Metabolism, Molnahl, Sweden (K.J. H.)

ABSTRACT A reversed-phase coupled column separation (CCS) system for the analysis of two diastereomeric glucuronides of almokalant, a new class I11 antiarrhythmic drug, in human urine is described. After direct injection of urine samples (50 pl) the glucuronides were isolated by complex formation on a terbium(II1) loaded strong cation exchanger at alkaline pH. The solutes were eluted from the precolumn by an acidic mobile phase, enriched and separated on Hypercarb (porous graphitic carbon) as analytical column with 0.1 M acetic acid pH 2.8 and 30% acetonitrile as mobile phase. The calibration graph was linear (r2 = 0.9999) and the detection limits were in the low picomole (W)or femtomole (fluorescence)range. Optimization of the analytical column revealed that elution order and selectivity for the glucuronides were dependent on the buffer agent and temperature used. By appropriate choice of mobile phase conditions all four diastereomers could be separated. o 1992 WiIey-Liss, Inc. KEY WORDS: bioanalysis, almokalant glucuronides, diastereomers,metal complexes, porous graphitic carbon INTRODUCTION

Almokalant (H 234/09; 4-[3-[ethyl[3-(propylsulfinyl)propyl]amino]-2-hydroxy-propoxy]benzonitrile,see Fig. 1) is a new class 111 antiarrhythmic agent which has been shown to induce selectively prolonged action potential duration and positive inotropy in experimental animals, 1*2 humans, and isolated human ventricular muscle. The drug is currently being evaluated in phase I1 clinical trials to determine its efficiency and safety. In healthy male subjects, almokalant is well tolerated and the dose is completely absorbed from the gastrointestinal tract with a bioavailability of about 50%.5 More than 90% of the dose is eliminated by the kidneys within 0-24 h and about 10%of the oral dose is found as parent compound in urine. The major metabolite in man has been identified as the glucuronide of almokalant6 with the glucuronic acid attached to the hydroxyl group (see Fig. 1). Almokalant has two chiral centres and has been developed as a mixture of four stereoisomers. Consequently, after conjugation with P-1)-glucuronicacid, a mixture of four diastereoisomers is obtained. The polar and ionizable character of glucuronides makes their bioanalysis difficult. Reversed-phaseion-pair LC7 and ligand exchange chromatography8 have been utilized for direct injection of biological matrices. Furthermore, ionpairg and cation exchange chromatography have been employed for glucuronides that contain an amino group. The aim of the present study was to develop a selective method for the determination of two diastereomeric glucuronides of almokalant in human urine by means of a coupled column separation system with UV and fluorescence detection. o 1992 Wiley-Liss, Inc.

The main objective was to retain the metabolites from urine at alkaline pH by complex formation with terbium(II1) metal ions loaded onto a strong cation exchanger as precolumn.8 After this clean-up step, the polar, mitterionic glucuronides were to be transferred on-line to the analytical column for enrichment and separation. Furthermore, the influence of concentration and choice of buffer componentsas well as the temperature and content of organic modifier in the analytical mobile phase were investigated in order to optimize the separation order in an attempt to resolve all four diastereomeric glucuronides of almokalant. The stability of the coupled column separation system was evaluated with respect to precolumn lifetime and glucuronide retention times.

MATERIALS AND METHODS Instrumentation The LC system consisted of LKB 2150 HPLC pumps (Bromma, Sweden), Rheodyne 7125 injector, and Rheodyne 7000 switching valves with Tefzel alkaline resistant rotor seals, SpectroMonitor 111 LDC (Milton Roy, Florida) UV detector, Kipp & Zonen BD 40 recorder, Model Ls-4 fluorescence detector (Perkin-Elmer,Beaconsfield, U.K.), and Haake Fe (BerlinSteglitz, Germany) waterbath for thermostating the chromatographic systems. Received for publication May 28, 1992; accepted July 9, 1992. Address reprint requests to Kurt-JijrgenHoffmann, Astra Hassle Research L a b ratories, Department of Pharmacokinetics and Drug Metabolism, S-431 83, Molndal, Sweden.

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STEFANSSON AND HOFFMA"

4

HO

OH

S-Almokalant glucuronide

t J

Almokalant

F.R.G.). The Nucleosil column was packed into the stainless steel column with a slurry technique using methanol-2-propano1 (6:4 v/v) as suspension medium followed by washing with 200 ml of methanol. Prior to analysis the column was equilibrated with at least 500 ml of mobile phase. The volume of the mobile phase in the columns, VM, was determined by the front disturbance in the chromatograms on injection of water and UV detection at 248 nm. The capacity ratio, k', and the selectivity, ci, were calculated from k' = ( VR - VM)/VM and u = k ' 2 / k ' l , where VR is the retention volume for the peak maximum and k'l and P 2 are the capacity factors for the first and second peak. The mobile phase flow rate was 1.0 ml/min. Human Study

R-Almokalant glucuronide

Urine was obtained from a healthy volunteer. The study design has been reported previously. Briefly, 25.6 pmol(9 mg) of almokalant in a citric buffer (pH 5.0) solution was given orally and urine was collected quantitatively. Samples collected and 24-36 h were at 0-2,2-4,4-6,6-8,8-10,lO-12,12-24, stored at - 20°C before analysis.

I

OH

Fig. 1. Structure of almokalant and main metabolites found in human urine. The two chiral centres in almokalant are indicated by asterisks.

The pH measurements were made with an AG 9100 Metrohm 632 pH meter (Hernisau, Switzerland)with a Type 1014 glass pH electrode. Samples were filtered with Millex-GV Millipore 0.22 pm, 13-mm filter units. Millipore-qualitywater was used for all solutions. Chemicals, Chromatographic Phases, and Preparation

All chemicals were of analytical grade and used without further purification. The glucuronidesof almokalant were synthesized at the Department of Organic Chemistry at Astra Hassle AB (Molndal, Sweden). They were provided as separated (R)- and (S)-form as depicted in Figure l , but as racemic sulphoxide. The (R)-(R,S) and (S)-(R,S) mixtures will be referred to as R and S, respectively. The four peaks obtained in some of the systems will be referred to as R1, R2, S1, and S2 according to their elution order. The precolumn (21 x 4.6 mm i.d.) was a home-made stainless steel column with a polished inner surface equipped with Swagelock connectors and Altex 250/21 stainless steel frits. The column packing was Hitachi-Gel #3011-S 10-12 pm, a spherical and macroporous sulphonated polystyrene resin (kindly provided by Hitachi Corp., Tokyo, Japan). The cation exchanger was packed with water as slurry medium and then converted to the terbium(II1) form by a 500 p1 injection of Tb(C1)3 solution (2 M in water) using 0.3 M 2-(N-morpholino)ethanesulphonicacid (MES) pH 5.0 as mobile phase. The excess was removed by a 10 ml wash with the same mobile phase and the column was then equilibrated with 0.1 M NaOH as mobile phase. The analytical columns used were Hypercarb 100 x 4.6 mm i.d. (Shandon, Scientific, Runcorn, U.K.), PLRP-S 5 pm, 150 x 4.6 mm i.d. (Polymer Laboratories Ltd., Amherst, MA) and Nucleosil CIS5 pm, 50 x 4.6 mm i.d. (Machery-Nagel, Diiren,

Chromatographic System

The set-up of the bioanalytical coupled column separation (CCS) system is shown in Figure 2. The terbium(II1) loaded precolumn was equilibrated with 5 ml of mobile phase A (0.1 M NaOH at a flow of 1.0 ml/min). Prior to injection (50 p1) the samples were made alkaline (pH 13 with 1 M NaOH) and filtered over Millex-GV (0.22pm) 13-mm filter units. The time needed for the clean-up wash was determined as 10 min by injection of urine and washing with sodium hydroxide (eluate directed to waste) until the UV-signal at 248 nm (0.005 AUFS) returned to the baseline and the endogenous compounds were removed. Addition of 20% methanol to the mobile phase did not reduce the wash time needed, but the back pressure was increased after some injections of urine. Consequently, 0.1 M NaOH was chosen as mobile phase. The analytes were highly retained by strong complex formation with the metal ions. The glucuronideswere desorbed by introduction of an acidic mobile phase with pump B 0.3 M MES buffer pH 5.0 in 20% methanol at a flow rate of 1.0 ml/min, and transferred to the analytical column during 2.25-7.0 min by the switching valve (No. 4 in Fig. 2) in a peak volume of 4.75 ml. The metabolites were enriched by adsorption onto the strongly hydrophobic porous graphitic carbon phase and separated by switching back to pump C, 0.1 M acetic acid pH 2.8 and 30% acetonitrile at 1.0 ml/min, for the analytical column followed by UV detec-

Pump A

Pump C 5

3

Pump B

waste

Fig. 2. Schematic presentation of the coupled column system. (1)Switching valve for pump A and B to the precolumn; (2) injector; (3) precolumn; (4) switching valve; (5) analytical column; (6) UV or fluorescence detector.

DIASTEREOMERIC GLUCUROMDES OF ALMOKALANT

511

further improved the selectivity.With addition of methanol, the glucuronides eluted during 2.25 to 7.0 min in a peak volume of 4.75 ml. Acetonitrile (10-20%) on the other hand, caused severe peak tailing, possibly by changing the swelling characteristics of the cation exchanger. A lowering of pH drastically increased the retention of the solutes and, as a result, the elution of glucuronides was limited to pH between 5 and 8. Consequently, the enrichment step on the analytical column will be even more difficult since the analytes will be present as zwitterions. The precolumn was stable for 12-15 (6 columns studied) injections and the stability was measured by the recovery of Optimization of the Analytical Mobile Phase the glucuronides obtained from consecutive injections of spiked The choice of buffer agent in the analytical mobile phase was urine samples. The decrease in recovery might be due to two found to be of great importance for selectivity and elution order reasons: first, the column bed or the particles collapsed by the of the diastereomericglucuronides. Various buffers were stud- increased backpressure, or second, endogeneous and hydrophoied with respect to stability of retention times in the coupled bic compounds present in the urine adsorbed to the polystyrene column separation systems. Most of these studies were per- matrix and competitively decreased the adsorption of the formed without the switching valves displayed in Figure 2. The glucuronides on the precolumn during the elution step; hence, analytes were injected directly onto the analytical column, i.e., the retention decreased resulting in loss of the glucuronides an injection valve was installed instead of the switching valve during transfer to the analytical column. A new precolumn was No. 4. The parameters investigated were nature and concentra- installed on a daily basis or when the backpressure increased, typically to about 50 bar. A spiked urine sample was injected tion of buffer agents, organic modifiers, and temperature. as duplicate and the recovery obtained was taken as a correlaCalibration Graph tion factor for the slope of the new standard curve. The relative Stock solutions of the (R)- and (S)-diastereomericglucuro- standard deviation of the recovery between the different preconides were prepared by dissolving in water to a final concentra- lumns was =: 15% (n = 5) for both glucuronides. tion of 1.5 mM. They were added to a blank urine sample and Choice of Analytical Column these standards (concentration range 0.4-30 pg/ml) were The polar and zwitterionic almokalant glucuronides were treated as described above and the calibration graphs were transferred from the precolumn to the analytical column in a constructed by plotting the peak area versus the concentration volume of 4.75 ml(20% methanol in 0.3 M MES pH 5.0). The of the samples. highly hydrophobic porous graphitic carbon-Hypercarbwas found to enrich the glucuronides efficiently and subseRESULTS AND DISCUSSION quently separate them using 0.1 M acetic acid in 30% acetoniPrecolumn: Principle and Choice of Conditions trile as mobile phase. If a reversal in elution order is desirable, Glucuronides, sugars, and glycoconjugatesform very strong formic acid can be used as buffering agent (see “Optimization” complexes with terbium(III),electrostatically immobilized on a below). strong cation exchanger, at alkaline P H . ~ The complex formaIt was not possible to retain the glucuronides on Nucleosil tion is governed by the ionization of one or several of the c18 solid phase using 20 mMdodecylammoniumas counter ion hydroxyl groups on the carbohydrate moiety. The presence of in 20% methanol and 0.05 M MES buffer pH 5.0 as mobile an acidic group (carboxyl or phosphate) generally increases the phase. This was opposite to the results obtained in a previous stability of the complex. The solute ion, Sx‘, competes with study on glucuronides7 and demonstrated the difficulty of rehydroxide ions for complex formation with the metal ion, taining zwitterions as ion pairs. The hydrophobic PLW-S colR-Tb2+,according to a ligand exchange reaction: umn (macroporous copolymer of styrene and divinylbenzene) also failed to retain the solutes. KS R-TNOH - ) y + S x - + R-TbS(0H - ) y - x + xOH Sensitivity and Repeatability of the CCS System

tion at 248 nm. The precolumn was washed for another 5 min with the MES buffer before changing back to 0.1 M NaOH as mobile phase and reequilibrated for 5 min after which a new sample could be injected. MES buffer was adsorbed onto the Hypercarb phase during the transfer and in order to get reproducible solute retention (day-to-day or after a couple of hours without any switch of mobile phase from the precolumn) 10 ml of the precolumn mobile phase (pump B) was introduced to the analytical column before the first sample was injected.

where Ks is the apparent stability constant for the reaction. The precolumn has to be converted to its hydroxide form before injection of the solutes. Introduction of an acidic mobile phase (20% methanol in 0.3 M MES buffer pH 5.0) splits the metal complexes and the glucuronides elute from the precolumn. The zwitterionic MES buffer does not give complexes with the metal ionss in contrast to other common buffers and has to be used in order to avoid metal stripping of the precolumn. The high buffer capacity was used to speed the neutralization of terbium hydroxide and the elution of the solutes. During the elution step, the zwitterionic glucuronides of almokalant were retained by hydrophobic interaction with the polystyrene matrix of the precolumn which

The standard curve for 02)-and (S)-glucuronidesbased on peak area measurements of spiked urine samples gave good linearity (9= 0.9999) and negligible intercept. The equation of the line wasy = - lo00 + 8009x for both metabolites (where y was area units and x was the concentration of the glucuronides in pg/ml). The relative standard deviation (repeatability) was 1.6% for S (n = 5; 5 pg/ml) and 2.1% and 4.0% for R and S (n = 10; 0.3 pg/ml), respectively. The detection limits (S/N = 3) for R and S were 10 and 16 pmol. The sensitivity could be improved 40-fold (250 and 400 fmol) when fluorescence detection (exc = 243 and em = 300 nm) was employed. However, a late eluting (k’ = 55) fluorescent impurity from the MES buffer prolonged the analysis time.

512

STEFANSSON AND HOFFMANN

Most of the glucuronides were found in the first collection interval, indicating rapid renal elimination of the metabolites. The total amount excreted was 13.7 pmol(54% of the administered dose) with an S/R ratio of 1.47 which indicated stereoselective formation and/or elimination of the diastereomeric almokalant glucuronides. Stereochemical considerations in glucuronic acid conjugation have been reported for, e.g., propranolol, a drug which exhibits stereoselective glucuronidation in dogs with S/R ratio of 3.5." Structurally, almokalant and propranolol have the aminopropanol moiety in common and the same type of glucuronide is formed. Stereoselectiveeffects in the glucuronidation of oxazepam in vitro and in vivo have been studied l2 as well as the regioselectivity associated with the conjugation of morphine enantiomers. l3

10

30 rnin

20

Fig. 3. Separation of the (R)- and (S)-diastereomericalmokalant glucuronide pairs from direct injection of 50 p1 urine. (A) 30 pg/ml, (B) 0.5 pg/ml, and (C) urine blank. UV detection at 248 nm. The mobile phase on the Hypercarb column was 0.1 M acetic acid in 30% acetonitrile. The given retention time refers to injection onto the precolumn.

Urinary Excretion of Almokalant Glucuronides Almokalant (9.0 mg) was given orally to a healthy male volunteer and chromatograms obtained after direct injections of 50 p1 urine are shown in Figure 3. The almokalant (R)- and (S)-glucuronideswere well separated with 0.1 M acetic acid in 30% acetonitrile as mobile phase but the peaks were broad and asymmetric due to partial resolution of the four diastereomers. Consequently, they were measured by integration of the peak area. After hundreds of injections, no changes in peak characteristics on the analytical Hypercarb column performance were observed. However, the precolumn had to be changed every day or in case of increased backpressure. The time profile of metabolite excretion is given in Table 1. TABLE 1. Urinary excretion of almokalant glucuronidesa Sample intervals

01) 0-2 2-4 4-6 6-8 8-10 10-12 12-24 24-36 0-36

Optimization of R l , R2,S l , and S2 Separation: Character and Concentration of Buffer Organic, sulphuric, and hydrochloric acids were used as buffer agents in order to prevent insoluble salt formation or precipitation on the carbon phase. The influence of buffer acid strength and hydrophobicity on the retention and selectivity of the R2 and S2 diastereomersis shown in Table 2. Generally,the retention increased with increasing concentration and hydrophobicity of the anionic buffer component, i.e., hydrochloric acid-acetic acid and acetic acid-propionic acid. The pKa values are 4.8 for the organic acids (except for formic acid 3.8) and below 0 for the inorganic acids. The second ionization constant of sulphuric acid is pKa = 2.0. The organic acid buffers were used at a concentration of 0.1 M at pH 2.8 in 30% acetonitrile and the concentration of negatively charged buffer components, acting as counterions for the cationic glucuronides were 0.001 M except for formic acid (0.01 M). Hydrochloric and sulphuric acid are completely ionized (about 2.8 mM of the sulphuric acid is present as divalent anion) and the retention and the selectivity were similar to the formic acid system. Consequently, the concentration of anionically charged buffer components, acting as counterions, governed the retention of the glucuronides. The influence of formic acid concentration on retention and selectivity is presented in Table 3. Both retention and selectivity (a)increased with increasing buffer concentration further illustrating the operative ion pair mechanism. The retention of the solutes will increase with increasing hydrophobicity of the anionic buffer component. Lower retention and change in elution order were, however, obtained (Table 2) within the organic acid series indicating the presence

pmol excreted

R

S

3.319 1.071 0.351 0.213 0.166 0.072 0.204 0.138 5.53

5.104 1.578 0.509

0.288 0.222 0.094 0.188 0.184 8.17

TABLE 2. Effect of buffers on retention and selectivitya

HC1 &SO4

Formate Acetate Propionate Butyrate

0.01 0.01 0.10 0.10 0.10 0.10

33.3 32.6 30.4 5.20 12.1 6.36

21.5 21.4 20.3 9.30 14.1 11.0

'Solid phase: Hypercarb. Mobile phase: buffer in 30% acetonitrile. "Dose: 25.9 pmol (9 mg) given orally to a healthy volunteer.

ha

= k'(RZ)/k'(%y

1.55 1.52 1.50 0.56 0.86 0.58

513

DIASTEREOMERIC GLUCURONIDES OF ALMOKALANT

TABLE 3. Effect of formate concentration on retention and selectivity“

13.1 15.8 17.3 18.9 22.4

0.01 0.02 0.05 0.10 0.20

13.8 16.9 18.4 20.3 23.9

14.9 21.7 26.5 29.4 34.9

14.9 21.7 26.5 30.4 36.4

1.08 1.28 1.44 1.50 1.52

“Solid phase: Hypercarb. Mobile phase: formate in 30% acetonitrile. *a = P(RZ)/P,9).

of more than one retention mechanism. The reversal in elution order might be explained by the degree of ionization and hydrophobicity of the buffer (compare formic and acetic acid). Most likely, the uncharged form of the buffer agent competed with the solutes for adsorption to the solid phase. This assumption was supported by the long equilibration times required, when the concentration of the buffer in the mobile phase, or the temperature were changed. Generally, 6 to 8 h were needed for equilibration of the systems and the time increased with increasing hydrophobicity of the mobile phase components. This was in contrast to the fast equilibration observed with Hypera r b in normal phase mode. l4 A separation of all four diastereomers was achieved with the hydrochloric, sulphuric and formic acid systems at the expence of analysis time. The citric, hydrochloric, and sulphuric acid systems gave unstable retention times in the CCS systems due to adsorption of MES buffer to the carbon phase. At these acidic pH, MES was probably retained as ion pair with the anionically charged buffer components.

extent with D-camphorsulphonic acid and L-tartaric acid as with the hydrochloric acid system and all peaks were partially resolved. Total resolution would, probably, have been achieved by a decrease of the acetonitrile content. This possibility was, however, not investigated. The peaks more or less coeluted in the L-ZGPand L-proline systems. In water-based systems, the use of specific hydrogen-bondinginteractions is probably limited because of competition from water. Effectsof Temperature The influence of temperature (12-59°C) on retention, k‘, and selectivity, ct,was studied with 0.01 M H2S04 in 30% acetonitrile as mobile phase (Fig. 4A and B). A maximum in retention was obtained, at 18°C (A) whereas the selectivity continuously increased with decreasing temperature (B) but with very different slopes for the two diastereomeric (R and S) pairs of the almokalant glucuronides. The temperature dependence on retention and selectivity indicated the presence of more than one retention mechanism. The impact on the selectivity was rather unexpected, assuming that the solid phase was homogeneous and strongly apolar. Hypercarb has been shown l5 to separate isomers efficiently and, predictably, the distribution to the flat and hydrophobic porous graphitic carbon is governed by the conformation and the ability of the solutes to expose hydrophobic parts of the molecule to the surface. The remarkably slow equilibration of the system (6-8 h), upon temperature change, indicated that the distribution of the mobile phase components was greatly influenced by temperature and, hence, the competition for adsorption sites on the solid phase. Separation of R1,R2,S l , and S2 From Urine

The four (R)- and (S)-diastereomericglucuronides were separated on the Hypercarb column using 0.01 M H2S04 in 25% acetonitrile as mobile phase (11°C) and fluorescence detection Chiral Buffers (Fig. 5). The number of theoretical plates and the assymetry The aim was to investigate ion pair retention of glucuronides factors were in the order of 4,000and 1.1, respectively, which by the use of chiral and anionic buffer agents in attempt to to the best of our knowledge are some of the best reported in utilize the possibilities of stereospecific interactions, preferen- the literature on Hypercarb.l6 The (R)- and (S)-glucuronides tially hydrogen bonding. The systems were 5 mM D-( +)-cam- had to be isolated from a pooled urine sample (0-36 h) using phorsulphonic acid in 30% ACN, 10 mM L-tartaric acid in the CCS system described in “Urinary Excretion” above, be34% ACN, 10 m M L-proline in 34% ACN, and 2 m M N- cause the MES buffer gave unstable retention times in the CCS benzoxycarbonylglycyl-L-proline(L-ZGP)in 1.5mM NaOH and systems. The glucuronides were collected from the eluate, 34% ACN. The glucuronides were retained to about the same evaporated under reduced pressure, dissolved in the mobile

40 1

k’

-

30

_ I

20

a R2

1.08

h\

’ I

1,06

R1

s2

1,04

s1

1$2

I A 10 I 10

I

30 40 Temperature Co

20

8

I

50

60

1.00

10

20 30 40 Temperature C‘

Fig. 4. Effect of temperature on (A) retention and (B) selectivityfor the glucuronides. The analytical mobile phase was 30% acetonitrile in 0.01 M sulphuric acid with Hypercarb as column.

50

60

514

STEFANSSON AND HOFFMAN

LITERATURE CITED 4 3

80 min

2 1

40

0

Fig. 5. Chromatogram of the four diastereomeric glucuronides of almokalant: 1 = S1,2 = S2,3 = R1, and 4 = R2, present in a pooled urine sample (0-36 h after administration). The glucuronides were first separated in the CCS system (see Fig. 3) and the eluate was collected and evaporated under reduced pressure. The glucuronides were dissolved in the mobile phase: 0.01 M H2W4 in 25% acetonitrile at l l T , separated on Hypercarb and detected by fluorescence (ex = 243 and em = 300).

phase, and injected. The individual amounts excreted were R1 = 2.19, R2 = 3.42, S1 = 4.52, and S2 = 3.56 pmol, respectively, when 25.6 pmol of almokalant was administered orally. Similar ratios were obtained between the metabolites with the urine excreted during the first 2 h. Interestingly, the (R)- and (S)-form of the chiral sulphoxide, located on the flexible side chain apart from the hydroxyl group that is conjugated, influenced the glucuronidation of almokalant and/or elimination of the metabolites to a different extent. CONCLUSIONS

The two diastereomers of almokalant glucuronides attached to the chiral hydroxylic group were determined by direct injection of urine samples at alkaline pH in a coupled column separation system. The system consisted of a polystyrene-based strong cation exchanger in terbium(II1) form as precolumn and porous graphitic carbon-Hypercarb-as analytical column. The stability and the selectivity on the separation column were strongly influenced by mobile phase additives and the analytes were retained by at least two different retention mechanisms. The calibration graphs showed excellent linearity and the sensitivity of the system was in the low picomole range, using UV detection.Following optimization of mobile phase and temperature using the Hypercarb column, all four diastereomers could be separated. In order to use this system for bioanalytical work, however, the glucuronideshad to be isolated off-line by the CCS method.

1. Carlsson, L., Almgren, O., Duker, G. Electrophysical and hemodynamic evaluation of a novel class 111 antiarrhythmic agent, H 23/09, in the anesthetized dogcomparison with quinidine and o-satolol. Eur. Heart J. ll(suppl):P2355, 1990. 2. Duker, G. D., Almgren, 0. H 234p9-a new, potent class 111 antianythmic agent. J. Mol. Cell. Cardiol. 22(suppl III):S82, 1990. 3. D W , B., Almgren, O., Bergstrand, R., Gottfridsson, C., Sandstedt, B., Edvardsson, N. Class 111 mode of action of H 23/09-a double-blind, crmover, study using transesophageal atrial stimulation in healthy volunteers. Cardiovasc. Drugs Ther. 5(suppl 3):364, 1991. 4. Carlsson, L., Abrahamsson. C., Almgren, 0.. Lundberg, C., Duker, G. Prolonged action potential duration and positive inotropy induced by the novel class 111 antiarythmic agent H 23/09 (almokalant) in isolated human ventricular muscle. J. Cardiovasc. Pharmacol. 18882, 1991. 5. Btitirnhielm, C., Almgren, O., Bergstrand, R., Lagerstrom, P-O., Re&dh, C.G. Twentieth Annual ACCP Meeting, Abstract 30, Oct. 13-16, 1991, Atlanta, Georgia. J. Clin. Pharmacol. 31:841, 1991. 6. Hoffmann, K-J., et al. Manuscript in preparation. 7. Stefansson, M., Westerlund, D. Reversed-phase ion-pair liquid chromatography of glucuronides. Retention and selectivity. J. Chromatogr. 499:411-421, 1990. 8. Stefansson, M., Westerlund, D. Selective isolation of glucuronides and carbohydrates by liquid chromatography as complexes with metal ions in alkaline solution. Chromatographia, submitted. 9. Svensson. J-O., Rane, A,, %we, J., Sjiiqvist, F. Determination of morphine, morphine-3-glucuronideand (tentatively) morphine-6-glucuronidein plasma and urine using ion-pair high-performance liquid chromatography. J. Chromatogr. Biomed. Appl. 230 427-432, 1982. 10. Piotrovski, V.K., Metelitsa, V.I. Ion-exchange high-performance liquid chromatography in drug assay in biological fluids. J. Chromatogr. Biomed. Appl. 231205, 1982. 11. Walle, T., Wilson, M., Walle, K., Stephen, A., Stereochemicalcomposition of propranolol metabolites in the dog using stable isotope-labeled pseudoracemates. Drug Metab. Disp. 11:544. 1983. 12. Sisenwine, S., Tio, C., Hadley, F., Lin, A,, Kimmel, H., and Ruelius, H.S., Species-related differences in the stereoselective glucuronidation of oxazepam. Drug Metab. Disp. 10605, 1982. 13. Rane, A,, Gawronska-Szklarz, B., Svensson, J. Natural ( - )- and unnatural (+)-enantiomers of morphine: Comparative metabolism and effect of morphine and phenobarbital treatment. J. Pharmacol. Exp. Ther. 234:761,1985. 14. Karlsson. A,, Pettersson, C. Enantiomeric separation of amines using Nbenzoxycarbonylglycyl-L-prolineas chiral additive and porous graphitic carbon as solid phase. J. Chromatogr. 543287, 1991. 15. Knox, J. H., Kaur, B. In: High Performance Liquid Chromatography. Brown, P. R., Hartwick, R. A. ed. New York Wiley, 1989: 189. 16. Lim, Chang-Kee Porous graphitic carbon in biomedical applications. In: Advances in Chromatography, Vol. 32, Giddings, J. C., E. Grushka, E., Brown, P. R. eds. New York: Marcel Dekker, 1992 1-19.