A New Bioanalytical Tool for Metabolite Analysis and Purification: OPLC Combined with Off-line and On-Line Radioactivity Detection Imre Klebovich1, Michel Manach2, Nathan Bryson2, William Amoyal2, Helmer Korb2, Emil Mincsovics3 1) Department of Pharmacokinetics, EGIS Pharmaceuticals Co. Ltd., Keresztúri út 30-38., H-1106 Budapest, Hungary., 2) Bionisis SA., 18-20 av. Edouard Herriot, F-92350 Le Plessis Robinson, France. 3) OPLC-NIT Ltd., Andor u. 60., H-1119 Budapest, Hungary
MATERIALS AND METHODS OSU 50 Purification Unit
OPLC 50
SCHEME OF OPLC SEPARATION USING OFF-LINE (A A) AND ON-LINE (B B) RADIOACTIVE DETECTIONS
ON-LINE NP-OPLC-RD RADIOCHROMATOGRAM OF GTN AND ITS METABOLITE STANDARDS ON HTSorbTM SILICA
OFF-LINE OPLC-DAR METABOLITE PROFILE (A A) AND RADIODENSITOGRAM PROFILE (B B) OF 3H-XXX COMPOUND METABOLITES IN DOG PLASMA AFTER 10 MG/KG ORAL ADMINISTRATION
14C
7,80
CP S *1 00 0
ON-LINE OPLC-RD AND OFF-LINE OPLC-DAR SEPARATION OF 14C-LABELLED METABOLITES IN AN URINE SAMPLE
3, 5 0
Front off-line radioactive detection
2, 5 0
2, 0 0
OPLC-DAR
1.2-GDN
1.3-GDN
GMN
1, 5 0
18,43
A
GTN
3, 0 0
OUTLET
1, 0 0
35,23 35,97
0, 5 0
Start
sample INLET
0, 0 0 0
5
10
15
20
25
30
35
min
time
ON-LINE NP-OPLC-RD CHROMATOGRAM OF GTN AND ITS METABOLITES ISOLATED FROM RAT PLASMA 15 MINUTES AFTER TREATMENT WITH GTN
Off-line OPLC
A
INTRODUCTION
METABOLITE STRUCTURE DETERMINATION (DIFFERENT SPECTROSCOPIC METHODS, e.g. MS, NMR)
18,08
8,48
OPLC-DAR
off-line radioactve detection
5 4 3
0
5
10
15
20
25
30
35
Exposition time: 4 hours
40
min
The biological sample can be purified by NP-OPLC and the metabolite fractions can be collected on the basis of on-line detected radioactive signal. The collected fractions can be further separated by HPLC-RD thus exploiting the high resolution capability of HPLC. For the detection of radionuclides, the combination of OPLC and digital autoradiography (DAR) and/or phosphor imaging techniques (PIT) has proved to be a powerful method in metabolite research [5, 6, 9].
DIRECTION OF DEVELOPMENT
GTN 1,3-GDN
COMPARISON OF OPERATING CONDITIONS FOR OPLC SEPARATION USING ON-LINE AND OFF-LINE RADIOACTIVITY DETECTION FOR 3H- OR 14C-XXX DRUG CANDIDATE
GMN START
PARAMETERS
ON-LINE PROCESS
OPLC 50 Bionisis SA, France
ALYTICAL INSTRUMENT:
Separation Rapid period Time of separation Flow rate External pressure Detection
OFF-LINE PROCESS
Mobile phase: acetonitril: n-dibutylether -A: 0:1 (v/v) -B: 1:9 (v/v) -C: 1:1 (v/v) Three-step-gradient 400 µl n-dibutylether Gradient: A: 26 min B: 2 min C: 9 min 1200 µl/min 5 MPa RD
Isocratic 400 µl n-dibutylether Isocratic: 6.1 min 600 µl/min 5 MPa DAR and/or Phosphor Imaging
REAL TIME DETECTION FROM CHROMATOPLATE: MEASUREMENT TIME:
Berthold LB287 (Wildbad, Germany)
BAS 1800 (Raytest Ltd., Straubenhardt, Germany) Sensitive area: 620 cm2 Imaging plate + casette: BAS-MS 2325 Film: FUJI BAS 1800 II. Software: BAS Reader ver: 3.01 AIDA ver: 3.11
Yes
No
4 hours
60 hours
Mobile phase: -A: chloroform :acetonitrile 40:60 (v/v) -B: 1-butanol:acetic acid:water 4:1:1 (v/v)
Separation Rapid period Volume of eluent
Stepwise gradient 300 µl eluent A Gradient: A: 100 µl B: 3700 µl
Flow rate External pressure DETECTION:
250 µl/min 5 MPa DAR; Run time: 120 min
PARAMETERS
ANALYTICAL INSTRUMENT: • Columns - guard - analytical • Mobile phase
3. B. Dalmadi Kiss, E. Mincsovics, K. Balogh Nemes, I. Klebovich. J. Planar Chromatogr., 13, 257-260 (2000)
4. E. Mincsovics, B. Dalmadi Kiss, Gy. Morovján, K. Balogh Nemes, I. Klebovich., J. Planar Chromatogr., 14, 312-317 (2001)
200 µl/min 5 MPa RD; Solide scintillator packed ce
• Gradient elution
C-GTN Separation in rat plasma
14
C-XXX Separation in human urine
Agilent 1100 (Waldbronn, Germany) LiChroCART 4-4 Purospher RP-18 endcapped (5 µm) LiChroCART 250-4 Purospher RP-18 (5 µm) A: 15 mM ammonium formate solution A: 15 mM ammonium acetate solution (pH 2.75) (pH 2.75) B: acetonitrile B: acetonitrile Linear: 5-80 % B (30 min)
• Flow rate • Temperature of column • Injected volume DETECTION: • Detector cell • Data acquisition
ANIMAL AND/OR HUMAN EXPERIMENTS (PLASMA, URINE, FACES, HEPATOCITES, MICROSOMES, etc.)
Stepwise gradient 400 µl eluent 250 µl
OFF-LINE NP-OPLC CHROMATOGRAM OF GTN AND ITS METABOLITES ISOLATED FROM RAT PLASMA USING PHOSPHOR IMAGING (PIT PIT) AS RADIODETECTION TECHNIQUE
Exposition Exposition time: time: 60 hours
FRONT
4 hours
CONDITIONS 14
5. I. Klebovich, Application of Planar Chromatography and Digital Autoradiography in Metabolic Research, In: Planar Chromatography, A Retrospective View for the Third Millenium, Ed. Sz. Nyiredy, Springer Scientific Publisher, Budapest, pp. 293-311 (2001)
7. E. Mincsovics, M. Manach, L. Kecskés, D. Papillard, E. Tyihák, J. Liqv. Chromatogr. Rel. Technol., 26, 2593-2609 (2003)
6. I. Hazai, I. Klebovich, Thin-layer Radiochromatography. In: Handbook of Thin-layer Chromatography, Third Edition. Eds.: J. Sherma, B. Fried, Marcel Dekker, Inc., New York, pp. 339-360 (2002)
8. D. Papillard, S. Laroche, E. Mincsovics, N. Bryson, LC GC EUR, Suppl. Appl. Book, April, 20-22 (2003)
Gradient profile:
5% B 3 min 5-17% B 12 min 17-30% B 10 min 30-60% B 15 min 60-100% B 2 min 100% B 8 min
1 ml/min 30 °C 100 µl On-line Radioactivity Detection (RD) Berthold LB 506-C-1 (Berhold, Wildbad, Germany) Solid Scintillator Packed Cell YG-150 U4D (Berthold) Gina Star ver. 2.03 (Raytest Ltd, Staubenhardt, Germany)
9. Gy. Morovján, B. Dalmadi Kiss, I. Klebovich, E. Mincsovics, J. Chromatogr. Sci., 40, 603-608 (2003)
COMPARISON OF VARIOUS CHROMATOGRAPHIC TECHNIQUES FOR ON-LINE OR OFF-LINE SEPARATION AND DETECTION OF RADIONUCLIDES IN METABOLISM RESEARCH FEATURE
Mobile phase - 1-butanol:acetic acid:water 4:1:1 (v/v)
SUMMARY OF OPERATING CONDITIONS FOR RP-HPLC SEPARATION USING ON-LINE RADIOACTIVITY DETECTION
OPERATIONAL CONDITION PHOSPHOR IMAGING
Sensitive area: 400 cm2 Multiwire proportional chamber: (MWPC) 360,000 detector cells High voltage: 14C 1200 V 3 H 2000 V Counting gas: argon-methane (9:1), 3.4 °C Software: WinDAR 1.09
for 14C-labelled human urine OPLC 50 Bionisis SA, France
DEVELOPMENT: Composition of OPLC eluent
Mobile phase: n-dibutylether 3600 µl
1 min 5 min 15 min 2 hours 4 hours Blood sampling time
ON-LINE PROCESS
OFF-LINE PROCESS for 3H-labelled dog plasma
ANALYTICAL INSTRUMENT:
OPERATING CONDITIONS OF THE APPLIED OFF-LINE RADIOACTIVITY DETECTION METHODS
MEASUREMENT CONDITION:
CONDITIONS OF SEPARATION AND RADIOACTIVITY DETECTION
PARAMETERS
conditions and on-line detection mode, can be estimated.
FRONT
1,2-GDN
CONDITIONS OF SEPARATION AND RADIOACTIVITY DETECTION
OPLC coupled with on-line radioactive detection (RD) or off-line DAR and/or phosphor imaging techniques (PIT) is an effective technique for the development and optimization of liquid chromatographic isolation, purifications and assay methods and could be also used for performing such micropreparative activities. For difficult separations, it can be easily combined with other chromatographic techniques, such as high-performance liquid chromatography (HPLC) on the analytical or preparative scale. Subsequent application of separation system with different selectivity may result in multidimensional separations. After OPLC separation in off-line mode (OPLC-DAR/PIT), the retention time of the compounds eluted from the layer under overrun
0
100-500 µl analytical layer (by spray technique) 1 cm off-line; 17 cm on-line 3 cm
COMPARISON OF OPERATING CONDITIONS FOR OPLC SEPARATION USING ON-LINE AND OFF-LINE RADIOACTIVITY DETECTION FOR TNG SEPARATION IN RAT PLASMA
CONCLUSIONS
2 1
Pre-washed Linomat IV (Camag, Switzerland)
• Band • Start position on layer
OPTIMIZED ISOCRATIC RP-HPLC ISOLATION OF THE ON-LINE OPLC COLLECTED PEAK (*)
7 6
CONDITION
(sealed on four sides)
• Volume
33,58
ON-LINE Distance on the layer
10 9 8
OFF-LINE NP-OPLC CHROMATOGRAM OF GLYCERYL TRINITRATE AND ITS METABOLITES ISOLATED FROM RAT PLASMA USING DIGITAL AUTORADIOGRAPHY AS RADIODETECTION TECHNIQUE.
20 x 20 cm 0.2 mm analytical layer Silica gel fine particle HTSorbTM
SAMPLE APPLICATION:
INSTRUMENT:
1. E. Tyihák, E. Mincsovics, Overpressured Layer Chromatography, In: Planar Chromatography, A Retrospective View for the Third Millenium, Ed. Sz. Nyíredy, Springer Scientific Publisher, Budapest, pp. 137176 (2001 2. J. Szúnyog, E. Mincsovics, I. Hazai, I. Klebovich, J. Planar Chromatogr., 11, 25-29 (1998)
time
HTSorbTM (Bionisis SA, France)
• Pre-conditions
DAR
BIOLOGICAL SAMPLE PROCESSING (PP; LLE; SPE)
OUTLET
OFF-LINE
PARAMETER
GMN
GTN
12 11
CONDITION OF OPLC LAYER AND SAMPLE APPLICATION
• Size • Thickness • Sorbent
1.2-GDN
15 14 13
Non-eluted compounds
PARAMETERS 1D & 2D SEPARATION OF METABOLITES AND CONCENTRATION (TLC-DAR; OPLC-DAR; OPLC-RD; HPLC-RD)
t0
sample INLET
LAYER:
14C
1.3-GDN
17 16
DIRECTION OF DEVELOPMENT
METABOLITE MAP
B
B
VELOPMENT: Composition of OPLC eluent
ROUTE OF METABOLISM STUDY
21 CPS 20 19 18
OPLC-RD
12,40
SCHEMES OF ONE (A A) AND FOUR-CHANNEL (B B) OPLC SEPARATION USING FLOWING ELUENT WALL FOR OPERATING SEGMENTATION AT INLET AND OUTLET.
10,97
on-line radioactive detection
For biotransformation investigations in vivo or in vitro, drugs labelled radioactively using 3H- or 14C- isotopes provide the possibility to track and quantitatively analyze the metabolites in complex biological matrices using separation techniques coupled to radioactivity detection methods. Although mass spectrometry can be powerfully used for qualitative analysis and structure elucidation combined with NMR, its application is difficult in the quantitative assay of an unknown metabolite since mass spectrometric response strongly depends on structure-related ionization efficiency. In contrast to mass spectrometry, radioactively labelled compounds will always have the same selective response on a radioactivity detection system. Although higher level of radioactivity provides higher response, practical reasons dictate that the activity of the radiolabelled substance should be reduced to the lowest amount that will provide adequate sensitivity. This also leads to the requirement to use instrumentation capable of detecting low intensity of radioactivity (minor metabolites). The objective of the present study was to evaluate the applicability of Optimum Performance Laminar ChromatographyTM (OPLC) [1] coupled to various on-line and/or off-line new radioactivity detection methods [2, 3, 4]. These detection methods include the on-line coupling of OPLC to a radioactivity detector equipped with a packed flow-cell with solid scintillator (OPLC-RD), detection of off-line separated compounds separated using digital autoradiography (DAR) or phosphor imaging (PIT) /bioimaging techniques. OPLC is a technique that uses a pressurized chamber, a pump system for the delivery of mobile phase into the chamber containing a flat chromatographic column under pressure. An external pressure of 50 bars is applied on the sorbent bed surface by means of e.g. a cushion system. Consequently the eluent is forced to flow through the sorbent bed [1, 5, 6]. For the comparison of the techniques, a pilot study on the identification and isolation of circulating metabolites of 14C-labelled glyceryl-trinitrate (GTN) and its metabolites (1.2-GDN, 1.3-GDN, GMN-s) and the new drug candidate of 3H- or 14C-labelled XXXcompound has been performed.
REFERENCES
RESULTS
10,72
A new simple and powerful bioanalytical solution was developed for the analysis, the isolation and the purification of minor and major metabolites in different biological matrices. The novel Optimum Performance Laminar ChromatographyTM (OPLC, Bionisis SA.) system equipped with the flowing eluent wall (FEW) technology is well adapted to off-line (eg. DAR) and on-line (RD) hyphenation with different radioactivity detectors. The simple purified biological sample can be applied for off-line or on-line OPLC separation. The eluted components from the HTSorbTM column can be detected by RD, while the non-eluted constituents can be evaluated by DAR. The newly developed on-line OPLC-RD method combined with off-line OPLC-DAR and/or OPLC-Phosphor Imaging (PIT)/Radioluminography and HPLC-RD techniques comprises a multidimensional rapid, economic and effective separation system for the study of in vitro and in vivo metabolism research. The purity of such isolated minor and major metabolites is appropriate for spectroscopic structure elucidation (eg. NMR, MS).
Distance on the layer
ABSTRACT
GTN 1,3-GDN 1,2-GDN GMN
START 1 min 5 min 15 min 2 hours 4 hours Blood sampling time
SEPARATION AND DETECTION TECHNIQUES ON-LINE PROCESS OFF-LINE PROCESS OPLC-RD HPLC-RD OPLC-DAR OPLC-PIT
SENSITIVITY SPEED RESOLUTION LINEARITY, RANGE QUANTITATIVE EVALUATION DETECTION LIMIT GLP CONFORMITY SAMPLE CAPACITY DETECTION OF DIFFERENT RADIONUCLIDES COST OF INSTRUMENTATION OPERATION COST
+++ +++ +++ +++ ++++ +++ ++++ ++++ ++++
+++ +++ ++++ +++ ++++ +++ ++++ ++ ++++
++++ +++ ++ ++++ ++++ ++++ ++++ +++ +++
+++ + ++++ ++++ ++++ ++++ +++ +++ ++++
++++ +++
++ ++
++++ ++++
++++ ++++
+ low/expensive, ++ good/fair, +++ high/acceptable, ++++ excellent/inexpensive
APPLICABILITY OF COMBINED TECHNIQUES IN METABOLISM RESEACH ASPECTS
OPLC-DAR/PIT
HPLC-RD
OPLC-RD
On-line, off-line operation Sample amount required Purity of separated and isolated metabolites Applicability for fast metabolite profile Applicability for fast fingerprint of conjugated metabolites Possibility of quantitative evaluation off over a wide linear range Suitability for preparation for structure elucidation Applicability for analysis of biological matrices with different radioactivity content Separation and isolation of metabolites when the signal/noise ratio is extremly low in biological matrix Cost effective metabolism study
off-line low medium yes (DAR yes (DAR)
on-line high high yes/no no
on-line medium high yes no
yes
yes
yes
MS/MS techniques
MS/MS, NMR techniques
MS/MS, NMR techniques
low radioactivity content animal, human study
high radioactivity content animal study
medium radioactivity content animal, human study
combination of methods yes
medium
medium
