Chapter 8
Developing Stereoselective High-Performance Liquid Chromatographic Assays for Pharmacokinetic Studies Irving W. Wainer Division of Pharmacokinetics, Department of Oncology, McGill University, Montreal, Quebec H4G 1A4, Canada
Enantioselective chromatography using H P L C chiral stationary phases (HPLC-CSPs) is rapidly becoming a stand procedure in analytical laboratories. This technique has been extensively used with pharmacologically acitve chiral compounds, although most of the reported separations are for the bulk drug substance. The application of H P L C -CSPs to pharmacokinetic and metabolic studies presents the analyst with addition problems often caused by interferrences from the matrix or from metabolites. One method to overcome these difficulties is coupled achiral/chiral chromatography which is discussed below. In the past few years there has been an increased interest in the pharmacological fate of chiral substances and a rapid growth in the study of the pharmacokinetic and metabolic disposition of stereoisomeric drugs. These development have been primarily due to the development and commercial availability o f chiral stationary phases for high performance liquid chromatography ( H P L C - C S P ' s ) . These phases have formed the backbone of numerous analytical methods capable of determining the in vivo concentrations of the various stereoisomers. Since the introduction in 1981 o f the (R)-N-(3,5-dinitrobenzoyl)phenylglycine C S P developed by W . H . Pirkle [7], the number of commercially available H P L C C S P s has grown to over 55. This wide variety of H P L C - C S P ' s has expanded the ability o f the analytical chemist to develop the necessary assays to follow the in vivo fate o f chiral drugs. It has also made it easier; for when there was only one or two H P L C - C S P ' s on the market it was often necessary to extensively alter the solutes to fit the requirements of the chiral recognition mechanisms operating on the C S P . This is no longer the case. With the large number o f available C S P s , a column can usually be picked to fit the properties of the solutes, eliminating the necessity for derivatization, and the requirements o f the assay. This situation is illustrated by the development of two assays for the determination of the serum concentrations o f the enantiomers of propranalol.
0097-6156/92/0512-0100S06.00/0 © 1992 American Chemical Society
8.
WAINER
Stereoselective HPLC Assays for Pharmacokinetic Studies 101
Serum Concentrations of Propranolol Enantiomers The first assay for the serum concentrations of the propranalol enantiomers using an HPLC-CSP was accomplished in 1983 using the (R)-N-(3,5-dinitrobenzoyl)phenylglycine CSP, DNPG-CSP, [2]. At that time, this was the only commercially available HPLC-CSP. In order to achieve an enantioselective separation on this CSP, the solute should contain one or more of the following functional groups: a ττ-acid or a ττ-base; a hydrogen bond donor and/or a hydrogen bond acceptor; an amide dipole [3]. The solute molecule should also not contain functional groups which are strongly cationic, i.e. a free amine moiety, or anionic, i.e. a carboxylic acid moiety [31 From the molecular structure of propranalol, it is clear that the substance had to be derivatized to eliminate the undesirable interactions caused by the free amine and to create additional positive interaction sites with the CSP. This was accom plished by reacting propranolol with phosgene to form an oxazoladone (Figure 1) which could then be chromatographed giving the separation illustrated in Figure 2. Since the assay had to be based upon the requirements of the CSP, the resulting procedures required the initial extraction of the target drugs from plasma using diethyl ether, addition of phosgene (12.5% solution in toluene), collection and evaporation of the ether layer, redissolution of the resulting solid in methylene, and injection onto the CSP. The observed retention times of the propranolol enantiomers were 4,839 and 5259 sec, Figure 2, making the assay both lengthy and complicated, especially since it employed a rather toxic derivatizing agent. Subsequent to the development and application of the assay utilizing the DNPG-CSP, a series of HPLC-CSPs based upon derivatized cellulose were developed; including a phase composed of cellulose tris(3,5dimethylphenylcarbamate), the OD-CSP [3], Using the OD-CSP, the enantiomers of propranalol could be enantioselectively resolved directly on the column without prior derivatization. This allowed for the development of a new assay which required only three steps: 1) the addition of sodium hydroxide to the serum; 2) extraction of the propranolol into an organic solvent; 3) injection of that extract onto the OD-CSP [4]. The resulting chromatograms are given in Figure 3. Thus the technological advancement of going from one column, the DNPG-CSP to the ODCSP, reduced both the preparation and analysis times as well as producing an assay which was more "user friendly".
The Need for Coupled Achiral/Chiral Chromatographic Systems As illustrated by the substitution of the OD-CSP for the DNPG-CSP, technological advances in the development of new CSP's has permitted the development of direct and simple assays. The drive of the analytical chemist is always to develop assays which consume the minimum amount of supplies (i.e. organic solvents, etc.) and technician time (i.e. extractions, derivatizations and length of chromatographic run). This had been made possible by the development of the wide variety of HPLC-CSPs. However, while the HPLC-CSPs are able stereochemically resolve the individual isomers of a chiral molecule, and at this stage it is safe to say that there are very few enantiomers which cannot be separated using this technology, they often cannot
CHROMATOGRAPHY OF PHARMACEUTICALS
102
R-CH-CHo
+
COClo 1
I I HO
HNCH(CH ) 3
2
R-CH-CHo / 0
\c/
\ ' N - C H ( C H T ; )2~
Figure 1. Synthesis of the oxazolidone of propranolol. See Reference 2 for experimental details.
Figure 2. Chromatogram of whole blood extract containing 50 ng racemic pro pranolol per ml. Peaks: A = oxazolidone corresponding to (S)-propranolol; Β = oxazolidone corresponding to (R)-propranolol; C = internal standard. For chromatographic conditions see Reference 2.
8.
WAINER
Stereoselective HPLC Assays for Pharmacokinetic Studies 103
differentiate between structurally related compounds. This is not a problem when you are dealing with the analysis of a chiral compound as a bulk drug substance or in a pharmaceutical formulation. However, it is a problem when you are dealing with drugs and their metabolites. This problem is illustrated by the example of verapamil (VER) and one of its major metabolites, norverapamil (NORVER). Determination of Verapamil and Norverapamil Enantiomers in Serum - First Approach. Using a CSP based upon immobilized a acid glycoprotein (AGP-CSP), VER and NORVER can be readily separated without derivatization, Figure 4A and 4B, respectively. However, when you chromatograph the parent, VER and the metabolite NORVER together, which would be the situation after administration of VER to a living organism, what you would find is that the compounds overlap as illustrated in Figure 4C. Thus, the AGP-CSP was able to stereochemically resolve the respective enantiomers of VER and NORVER, but unable to separate parent from metabolite. One approach to overcoming this problem of structural selectivity and to maximize the utility of the CSP's enantioselectivity is to use coupled achiral/chiral chromatography. In this approach an achiral column is used to separate the target compound or compounds from the biological matrix, as well as from each other. The eluents containing the target compound or compounds are then directed onto the CSP where they are stereochemically resolved. A diagram of one type of coupled achiral/chiral chromatographic system is presented in Figure 5. The system presented in Figure 5 has been used to solve the problems presented by VER and to analyze the enantiomeric compositions of both VER and NORVER in plasma [5]. In this assay, the achiral column contained a shielded hydrophobic phase (Hisep) which was able to separate VER from NORVER and both compounds from the other components in the plasma matrix. The eluents containing VER and NORVER were selectively transferred to the AGP-CSP where the enantiomers were stereochemically resolved and the enantiomeric composition determined. The resultant chromatograms from the AGP-CSP are presented in Figure 6. This assay has been validated and used extensively in single dose studies. However, while this is a successful application of the achiral/chiral coupled column system depicted in Figure 5, there are a number of problems which plague this type of coupled column system. For example, a single assay requires 2 injections -the first is used to quantitate the total drug on the achiral system and the eluent passes from the achiral column to the detector and then to waste; the second injection is used to provide the eluent which is switched to the CSP for enantiomeric analysis. This approach is not only cumbersome and time consuming, it is often insensitive on the chiral end of the analysis due to loss of compound during the switching process and to band broadening. While it is often impossible to avoid the type of coupled achiral/ chiral chromatography required for the separation of VER and NORVER on the AGP-CSP, the continuing development of new HPLC-CSPs often presents new possibilities. This can also be illustrated by the development of a new assay for the enantiomeric composition of VER and NORVER in plasma. r
Determination of Verapamil and Norverapamil Enantiomers in Serum - Second Approach. Okamoto, et al. [6] have recently reported the development of a new series of HPLC-CSPs based upon amylose, and in particular^ 3,5 dimethylphe-
104
CHROMATOGRAPHY OF PHARMACEUTICALS
Blank Serum
Serum 11 μ Standard
Subject Sample
-Li-
Figure 3. Representative chromatograms for: A = an extracted blank serum sample; Β = serum sample spiked with racemic propranolol (150 ng per ml); C = serum sample from a volunteer subject 12 h after ingestion of a 160-mg dose of racemic propranolol. See Reference 4 for chromatographic conditions.
Time (min)
Time (min)
Time (min)
Figure 4. Representative chromatograms from the chromatography of racemic verapamil (VER) and racemic norverapamil (NORVER) on the AGP-CSP. A = racemic VER; Β = racemic NORVER; C = 50:50 mixture, racemic VER and racemic NORVER. Peaks: 1 = (R)-VER; 2 = (S)-VER; 3 = (R)-NORVER; 4 = (S)-NORVER. See Reference 6 for chromatographic conditions.
8.
WAINER
Stereoselective HPLC Assays for Pharmacokinetic Studies
Pump 1
A c h i r α I
Injector 1
Ρ h α s e
Pump 2
C h i r a l
Column I
Ρ h a s e
Column 2
Ζ
Switching valve Detector I
Detector 2
Total concentration
Enantiomeric ratio
CR-isomer] « [Total] · R/S CS-isomer] »CTotal]-CR3
Figure 5. Schematic representation of a Coupled Achiral/Chiral Chromato graphic System.
1
106
CHROMATOGRAPHY OF PHARMACEUTICALS
nylcarbamate-derivatized amylose coated on aminopropyl silica, AD-CSP. The ADCSP is able to achieve the enantioselective resolution of underivatized VER and NORVER without overlap has allowed for the direct separation of verapamil and norverapamil without the necessity of column switching. However, when this column was applied to chronic dosing studies, it became evident that there were additional interferences with the chromatography of VER and NORVER from a variety of other VER metabolites which are listed in Table 1, [7]. As you can see from this table, on the AD-CSP, (R)-VER and metabolite D617 overlapped as did (S)-NORVER and metabolite PR25. The problem presented by the coelution of (R)-VER and D617 and (S)NORVER and PR25 was solved by another form of coupled column chromatography in which an achiral column containing a LiChrocart diol silica was placed in front of the AD-CSP and in series with the CSP [7]. This changed the retention times of the metabolites as well as VER and NORVER, Table 1, and allowed for the total separation of all the compounds. The resulting chromatogram is presented in Figure 7. TABLE 1. RETENTION TIMES OF VERAPAMIL AND ITS METABOLITES ON AN AD-CSP WITHOUT (A) AND WITH (B) A LICHROCART DIOL COLUMN COUPLED IN SERIES. Chromatographic conditions: mobile phase, hexane: propanol-2:ethanol (85:7.5:7.5, v/v/v) containing 1.0% triethylamine; flow rate, 1.0 ml/min; excitation, 272 nm; emission, 317 nm; temperature, ambient. See reference [7] for further details Compound
Retention time (min) Without DIOL With DIOL
(S)-Verapamil (R)-Verapamil (S)-Norverapamil (R)-Norverapamil D617 PR25 PR22 (1st isomer) PR22 (2nd isomer)
6.7 7.7 10.5 11.7 7.4 10.7 15.5 18.2
7.4 8.4 12.2 13.4 17.0 32.8 18.6 21.2
Sequential Achiral/Chiral Chromatography When there are many metabolites to deal with or when the biological matrix is extremely complicated the analytical chemist may be forced into another form of coupled column chromatography, sequential achiral/chiral chromatography. While this is in fact the easiest of the coupled column chromatographic methods to develop it is also the one which is the most time consuming. The approach is as follows: the compound or compounds of interest are separated first on an achiral column; the
8.
WAINER
107 Stereoselective HPLC Assays for Pharmacokinetic Studies
A
Β
C 2
3
10
Time (min)
20
20
10
Time (min)
10
20
Time (min)
Figure 6. Representative chromatograms from the chromatography of verapamil (VER) and norverapamil (NORVER) on the AGP-CSP coupled to the Hisep HPLC column. A = serum blank; Β = serum spiked with 100 ng/ml each of racemic VER and NORVER; C = serum sample taken 12 h after the adminis tration of a 240-mg sustained release dose of racemic VER. Peaks: 1 = (R)VER; 2 = (S)-VER; 3 = (R)-NORVER; 4 = (S)-NORVER. See Reference 6 for chromatographic conditions.
c υ
Figure 7. Representative chromatograms from the chromatography of verapamil (VER) and norverapamil (NORVER) in plasma on an HPLC system with a DIOL column (5 cm χ 4.0 mm I.D.) coupled in line to an AD-CSP (25 cm χ 4.6 mm I.D.). See Reference 7 for chromatographic conditions.
CHROMATOGRAPHY OF PHARMACEUTICALS
108
eluent containing these compounds are collected and concentrated; then reinjected onto the CSP. This approach is illustrated in Figure 8. The collection of the eluents and their concentration all take place off-line requiring additional labor in preparing the compounds for the second injection. A mixture which contains four target compounds will require a single injection to separate the compounds and four additional injections of each one of the isolated compounds; thus, 100 samples requires 500 injections. While this method is time consuming, it often the only way to solve the problem. This situation is illustrated by the assay for the enantiomeric composition of hydroxychloroquine and its major metabolites in plasma [#]. In this approach, hydroxychloroquine and its metabolites desethylchloroquine, desethylhydroxychloroquine and bidesethylchloroquine were first separated on a achiral column containing a cyano-bonded stationary phase. The eluents were collected and concentrated on a Speed-Vac evaporator and reconstituted in the mobile phase used on the AGP-CSP, the CSP used in this assay. Then each one of the compounds was injected on to the AGP-CSP. The chromatographic parameters for these separations are presented in Table 2.
TABLE 2. CHROMATOGRAPHIC PARAMETERS OF H Y D R O X Y C H L O R O Q U I N E (HCQ), BIDESETHYLCHLOROQUINE (BDCQ), DESETHYLHYDROXYCHLOROQUINE (DHCQ) AND DESETHYLCHLOROQUINE (DCQ) ON AN ACHIRAL CYANO-BONDED PHASE AND THE AGP-CSP. See reference [8] for further details
Cyano column .b k"
Compound
k
BDCQ DHCQ DCQ HCQ a
c
12.11 10.19 11.44 8.67
1.61 2.56 3.93 5.33
AGP-CSP « R c
d
RS
1.25 1.32 1.39 1.39
1.29 1.39 1.97 2.08
b
Capacity factor; Capacity factor first eluted enantiomer; Enantioselectivity factor; Enantiomeric resolution d
The sequential achiral/chiral assay for the enantiomeric concentrations of hydroxychloroquine and its enantiomers in plasma has been validated and is now in use in pharmacokinetic and metabolic studies.
8.
WAINER
Stereoselective HPLC Assays for Pharmacokinetic Studies 109
PLASMA EXTRACT REINJECT
A
C H I R A
L
1
c H I R A L
1
I lj I Jl
1 1
COLLECT AND CONCENTRATE
Figure 8. Schematic representation of a Sequentially Coupled Achiral/Chiral Chromatographic System.
110
CHROMATOGRAPHY OF PHARMACEUTICALS
Conclusion W h i l e the development o f pharmacokinetic and metabolic studies o f the enantiomeric compounds in biological fluids may at first seem difficult and time consuming, it is only because we are currently in a transition from the use of H P L C - C S P s solely for the separation of bulk drugs to their routine application in the bioanalytical laboratory. Within a few years this w i l l change and these phases w i l l be commonly found in the pharmacokinetic and bioanalytical laboratories. A l o n g with the standard use o f H P L C - C S P w i l l come column switching and similar techniques which are a normal part o f application of these C S P s . On the other hand, the development o f improved C S P s and the progress in enantioselective separations utilizing newer technologies such as capillary electrophoresis may make these approaches obsolete. What ever happens, there is an exciting and challenging future ahead for the separation o f enantiomeric compounds and the understanding o f their pharmacokinetic and metabolic fate.
Literature Cited [1] [2]
[3] [4] [5] [6] [7] [8]
Pirkle, W . H . , Finn, J . M . , Schriner, J . L . , Hamper, B . C . J. Am. Chem. Soc. 1981, 103, 3964-3966. Wainer, I.W., Doyle, T . D . , Donn, K.H., Powell, J.R. J. Chromatogr. 1984, 306, 405-411.
Wainer, I.W. A Practical Guide to the Selection and Use of HPLC Chiral Stationary Phases, J.T. Baker Inc., Phillipsburg, NJ, 1988. Straka, R.J., Lalonde, R . L . , Wainer, I.W. Pharm. Res. 1988, 5, 187-189. Chu, Y.-C., Wainer, I.W. J. Chromatogr. 1989, 497, 191-200. Okamoto, Y . , Aburatani, R., Fukumoto, T . , Hatada, K. Chem. Lett. 1987, 411-414. Shibukawa, Α . , Wainer, I.W. J. Chromatogr. 1992, 574, 85-92. Iredale, J., Wainer, I.W. J. Chromatogr. 1992, 573, 253-258.
RECEIVED May 5,
1992
