Oxidative Metabolism of Figure 10: Amodiaquine using the ... · The data for the MS Voltammogram were recorded using a scan mode with a potential range be-tween 0 and 1500mV, scanned

Oxidative Metabolism of Amodiaquine using the ROXY™ EC System

®

References1. Lohmann W., Baumann A., Karst U., Electrochemistry and

LC–MS for Metabolite Generation and Identifi cation: Tools,Technologies and Trends, LC•GC Europe Jan., (2010) 1-6.

2. Lohmann W., Hayen H., Karst U., Covalent ProteinModifi cation by Reactive Drug Metabolites UsingOnline Electrochemistry/Liquid Chromatography/MassSpectrometry, Anal. Chem.,80, 2008, 9714–9719

3. Lohmann W., Karst U., Generation and Identifi cationof Reactive Metabolites by Electrochemistry andImmobilized Enzymes Coupled On-Line to LiquidChromatography/Mass Spectrometry Anal. Chem. 79,2007, 6831-6839

4. Baumann A., Karst U., Online electrochemistry/massspectrometry in drug metablism studies: principlesand applications. Expert Opin. Drug Metab. Toxicol. 6,2010, 715

Figure 10: ROXY™ EC System consisting of ROXY Potentiostat, dual syringe

pump and ReactorCell.

Ordering number210.0070A ROXY™ EC system, incl. dual syringe pump, ReactorCell,

electrodes and LC connection kit for phase I and II reac-tions. All parts included for described Electrochemical (EC) application.

Electrifying Reactions Using EC/MS


Introduction Amodiaquine (AQ) is an antimalarial agent which is used against Plasmodium falciparum, a proto-zoan parasite which can cause cerebral malaria. Though the drug was withdrawn from the market because of its hepatotoxicity, it is still widely applied for the treatment of Malaria in Africa. Amo-diaquine is metabolized to reactive electrophilic metabolites, which are difficult to detect since they are shortlived, and the metabolites can undergo further reactions resulting in stable products.

Amodiaquine (trade names: Camoquin, Flavoquine; IUPAC: 4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)phenol) was chosen as a model drug to investigate the nature of the oxida-tive metabolism using the ROXY EC System.

Electrochemical conversion of the amodiaquine into reactive phase I metabolites and their GSH conjugates were successfully achieved.

■ Amodiaquine, Camoquin, Flavoquine

■ Fast mimicking and predicting drug metabolism < 10 min.

■ Oxidative metabolism (phase I) and adduct formation

(phase II)

■ Ideal for system performance evaluation

(reference system)

®Application Note

Drug Metabolism

Electrochemical Reactions upfront MS – EC/MS

Proteomics &Protein Chemistry

S-S bond reduction HDX

Peptide bond cleavageNa+, K+ removalDrug-protein binding

Lipidomics & Fatty AcidsCholesterolOxysterolFAME Biodiesel

Drug MetabolismMimicking CYP 450 Phase I & IIBiotransformation

Synthesis (mg) Metabolites & Degradants

Pharmaceutical StabilityPurposeful degradation API testing

Antioxidants

Environmental Degradation & persistenceTransformation productsSurface & drinking water

Food & BeveragesOxidative stability

Antioxidants

Forensic ToxicologyDesigner drugsIllicit drugs

Healthcare & CosmeticsSkin sensitizers

GenomicsDNA DamageAdduct formationNucleic acid oxidation

Conclusion

The on-line coupling of the ROXY™ EC System with MS (EC/MS) pro-vides a versatile and user-friendly platform for fast screening of target compounds (drugs, pharmaceuti-cals, pollutants, etc.) for oxidative metabolism (phase 1 reactions), thereby mimicking the metabolic pathway of CYP450 reactions.

MS voltammograms can be record-ed automatically to obtain a meta-bolic fi ngerprint of the compound of interest in less than 10 min.

In addition, rapid and easy studies of adduct formations can be per-formed simply by adding GSH after the ReactorCell (phase II reactions).

Figure 8: Result of conjugation of phase I metabolites of Amodiaquine with GSH. Example of EICs of Metabolite 1 (m/z 354) and its conjugate (m/z 661) and Metabolite 2 (m/z 326) and its conjugate (m/z 633)

Phase IITo confirm the presence of the conjugation products of Amo-diaquine metabolites and GSH, mass spectra were acquired with the ReactorCell off and at Ec = 400 mV and 1200 mV. EIC traces of Amodiaquine metabolites (1 and 2) are presented in Fig. 8. Mass spectra obtained with different potentials and a control experiment with ReactorCell OFF are shown in Fig. 9.

Figure 9: Mass Spectra of GSH-Metabolite adducts formed at 400 and 1200 mV with m/z 661.2 and 663.2, respectively. The spectrum with ReactorCell OFF confi rms that the conjugates are formed only if potential is applied.


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ApplicationNoteD_Amodiaquine_07 Drieslag.indd 4-6 30-09-14 11:13



Figure 1: Metabolic pathway of amodiaquine with the 3 most abundant metabolites.

Figure 2: Instrumental set-up of ROXY EC System for oxidative metabolism phase I.

Figure 3: Instrumental set-up of ROXY EC System for oxidative metabolismphase I.

Figure 4: Instrumental set-up of ROXY EC System generating the oxidative metabolites in the ReactorCell (phase I) and subsequnet addition of glu-tathione via a T-piece for GSH-adduct formation (phase II).

Method The ROXY EC System (Figure 2) for compound screening (p/n 210.0070A) includes the ROXY potentiostat equipped with a ReactorCell™, infusion pump and all necessary LC connec-tions. The ROXY EC System is controlled by Antec Dialogue software.

The ReactorCell equipped with Glassy Carbon working elec-trode and HyREF™ reference electrode was used for the gen-eration of amodiaquine metabolites.

The amodiaquine sample was delivered to the system with asyringe pump equipped with a 1000 μL gas tight syringe. A MicrOTOF-Q (Bruker Daltonik, Germany) with an Apollo II ion funnel electrospray source was used to record mass spectra and MS data were analyzed by Compass software. The rele-vant mass spectrometer parameters are listed in Table 3. The method was optimized on a 10μM amodiaquine solution. Mass spectrometer calibration was performed using sodium formate clusters at the beginning of the measurements.

Amodiaquine and its (selected) metabolites

Name Formula Monoisotopic mass [u]Amiodaquine (AQ) C20H22ClN3O 355.14514

1 (quinoneimine) C20H20ClN3O 353.12949

2 (desethyl; quinoneimine) C18H16ClN3O 325.09819

3 (bis desethyl; aldehyde) C16H11ClN2O2 298.05091

Table 1

Conditions

EC ROXY™ EC System (p/n 210.0070)

Cell ReactorCell™ with GC WE and HyREF™

Flow rate 10 μL/min

Potential 0 – 1500 mV (scan mode)

Mobile phase 20 mM ammonium formate (pH 7.4 ad-justed with ammonium hydroxide) with 50% acetonitrile

Conditions

Name FormulaMass range 50 – 1000 m/z

Ion polarity Positive

Capillary voltage -4500 V

Nebulizer 1.6 Bar

Dry gas 8 L/min

Temperature 200 C

ISCID energy 100 Vpp 0 eV

Hexapole 100 Vpp

Ion energy 5 eV

Table 2

Table 2

Oxidative metabolism – Phase IA 10μM amodiaquine solution in 20mM ammonium formate (pH 7.4 adjusted with ammonium hydroxide) with 50% ace-tonitrile was pumped at a constant flow rate of 10 μL/min through the ReactorCell using an infusion pump. The outlet of the reactor cell was connected directly (online) to the ESI-MS source. The scan mode was used to register the MS Voltam-mogram with the working electrode potential ramped from 0 – 1500 mV at a scan rate of 10 mV/s in the half cycle. The mass spectra for each change of the cell potential were recorded continuously and saved in one file. The total run time to re-cord the mass voltammogram was approximately 2.5 min. Instrumental set-up of ROXY EC System for oxidative metabo-lism phase I is shown in Figure 3.

Adduct formation – Phase IIA 10μM amodiaquine solution in 20mM ammonium formate (pH 7.4 adjusted with ammonium hydroxide solution) with 50% acetonitrile was pumped with a constant flow of 10 μL/min through the ReactorCell using an infusion pump. Adduct formation of amodiaquine metabolites and glutathione (GSH) was established using a 100μL reaction coil placed between the ReactorCell and the electrospray source. 100μM gluta-thione in mobile phase was added at the same flow rate via a T-piece into the coil and the reaction time at the specified flow rate was 5 min. The effluent from the reaction coil was injected directly into the ESI-MS. The instrumental set-up of the ROXY EC System for adduct formation (phase II reactions) is shown in Figure 4. The DC potentials of 400mV and 1200mV were applied to form conjugates with Metabolite 1, and Me-tabolites 2 and 3 (Fig. 1), respectively.

Results

Phase ITable 1 provides a list of compounds related to amodiaquine

metabolism and their monoisotopic masses used for mass

spectra interpretation. The 3-D MS Voltammogram shown for

amodiaquine (Fig. 5) is a graphical representation of oxidative

pattern of the analyte. The data for the MS Voltammogram

were recorded using a scan mode with a potential range be-

tween 0 and 1500mV, scanned at a 10mV/s rate in the half

cycle (Fig. 6).

The background information about MS Voltammogram acqui-

sition using Dialogue are given in the “Dialoque for ROXY user

guide” (P/N 210.7017) and in the application note 210_001A

“Event Programming for Automated Recording of MS Voltam-

mograms” for details, see our web.

The extracted ion chromatograms for the mass-to-charge ratio (m/z) of amodiaquine (m/z of 356) and its metabolites (m/z of 354; 326; 299 and 370) are shown in Figure 6 as a 2-D MS Voltammogram. Based on the 2-D MS Voltammogram (Fig. 6), the optimum potential for the formation of the particular metabolites was estimated as 400mV for amodiaquine dehy-drogenation (metabolite 1), and 1200mV for formation of me-tabolites 2, 3 and 4.

Furthermore if the potential is higher than 1400mV, hydroxyl-ation of Amodiaquine (m/z of 370) was observed. Fig. 7 shows the mass spectra corresponding to ReactorCell OFF (control measurement) with applied voltages of 400mV and 1200mV.

Figure 6: Amodiaquine abundance vs. EC potential. The 2-D MS Voltammo-gram was acquired using scan mode.

Figure 7: Mass spectra of phase I metabolites of Amodiaquine.

Figure 5: Mass voltammogram of Amodiaquine. Ion abundance versus m/z as a function of EC potential.


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Alexys Neurotransmitter Analyzer forMonoamines and Metabolites



Figure 1: ALEXYS Neurotransmitters Analyzer.

Figure 2: Analysis of monoamines and metabolites (standards) showing a separation within 2 (left) and a 5 µL injection of 100 pmol/L DA and 5HT in Ringer with 10 mmol/L HAc (right). A LOD of 50 pmol/L for DA and 65 pmol/L for 5HT is obtained. There is a trade-off in speed of analysis vs. de-tection limit and resolution in biological samples.

Figure 3: Schematic configuration of sampling 2 parallel systems with a 10-port valve.

Figure 4: Analysis of standards 5 nmol/L acidic metabolites and 0.5 nmol/L monoamines in Ringer solution with 10 mmol/L acetic acid.

Summary In this application note a fast and sensitive method is pre-sented for the analysis of monoamines and metabolites. A microbore UHPLC column is applied in combination with electrochemical detection using a high sensitivity wall jet flow cell. Detection limits down to 50 pmol/L have been achieved for dopamine. Analysis times vary between 1-15 minutes depending on the complexity of the sample matrix and the number of substances of interest. The system can be equipped with an additional channel for simultaneous analy-sis of (for example) the metabolites.

Method and resultsIn method development for the analysis of monoamines and metabolites a number of parameters are optimized to meet the requirements for detection limits, the use of small sam-ples, and short analysis times of multiple components.

Small sample volume and low detection limitsIn a previous communication the optimization for best pos-sible detection limit using small samples has been described [8]. Briefly, a wall-jet micro flow cell has been used which is fully compatible with microbore HPLC. It is well known that small samples are best analyzed using microbore LC with less peak dilution, resulting in more signal. Miniaturization using micro electrodes results in smaller noise. With this combina-tion an improved signal-to-noise ratio with detection limits down to 50 pmol/L (dopamine) have been achieved for the analysis of standards (Fig. 2).

The ALEXYS Neurotransmitter Analyzer consists of a DECADE II electrochemical detector, an OR 110 degasser unit and LC 110S pump(s), an AS 110S autosampler and Clarity data ac-quisition software. Complementary kits for analysis of nor-adrenaline, dopamine, serotonin and metabolites, GABA and glutamate or acetylcholine and choline are available.

Separation and speed of analysisTo meet the requirement for fast analysis of multiple compo-nents is a matter of selecting a suitable column and optimiz-ing the mobile phase. An analysis time less than 2 minutes is feasible for standards using UHPLC (Fig. 2), however we found there is a trade-off in analysis time vs. detection limit and res-olution when analyzing biological samples.

Speeding up a standard HPLC analysis with a factor 2 – 4 using UHPLC columns is feasible, depending on sample matrix and injection volume. At higher velocities a few things are limiting the performance. One of the most critical was the analysis of NA which is close to the solvent front (see figure 4). At high flow rates the peaks overlapped or disappeared in the front. Assumingly this is due to non-ideal behavior of high concen-tration matrix components eluting in the front peak.

The method and results presented describe an approach, not a fixed set of conditions. It is very well possible or even re-quired to tune the method for a different matrix composition (f.e. measuring in a different brain area) or a different set of neurotransmitters or metabolites of interest. In other words, the ALEXYS system is a flexible system which is not limited to a few applications. There is range of columns available to assist you in optimizing the chromatography to your specific application needs.

Secondary HPLC channel for metabolitesTo extend the possibilities a secondary channel can be add-ed to the ALEXYS Neurotransmitter system (Fig. 1). The dual channel system contains one additional pump, a column, and uses an autosampler with a 10 port valve and a dual channel detector.

Both channels share the same autosampler and electro-chemical detector (Fig. 3). Detection parameters and HPLC conditions are optimized for each channel depending on the substances of interest. Channel 1 is optimized for NA, DA and 5-HT. Channel 2 is for the acidic metabolites 5-hydroxyindole acetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA).

2 3

Conditions for analysis of monoamines (NA,DA, 5-HT) and metabolites

HPLC ALEXYS Neurotransmitter Analyzer

Oven temperature 38 °C (separation and detection)

Injection method 5 µL per column

ASSAY 1 (MPHG DOPAC, 5-HIAA, HVA)

Flow rate 100 µL/min, pressure about 420 bar

Flow cell Sencell 2 mm GC, sb, spacing 0.5

Column Acquity HSS T3 1 x100 mm, 1.8 um + pre-filter

ADF™ off

Range 10 nA/V

Ecell 800 mV

Icell 0.9 nA

Mobile phase 50 mM phosphoric acid, 0.1 mM EDTA, pH 3.0, 10 % ACN

ASSAY 2 (NA, DA and 5-HT)

Flow rate 100 µL/min, pressure about 420 bar

Flow cell Sencell 2 mm GC, sb, spacing 0.5

Column Acquity HSS T3 1.0x100 mm, 1.8 um

ADF™ Off

Range 1 nA/V

Ecell 460 mV

Icell 0.6 nA

Mobile phase 200 mM Acetic Acid, 0.1 mM EDTA, 300 mg/L DSA, pH 5.5, 14 % ACN

LOD: 80 pM NA (0.40 fmol), 70 pM DA (0.35 fmol), 100 pM 5-HT (0.50 fmol), 169 pM DOPAC (0.85 fmol), 69 pM 5-HIAA (0.34 fmol), 208 pM HVA (1.04 fmol)

4

Figure 5: Analysis from a single injection of basal level rat Prefrontal Cortex (PFC) microdialysate. In both chromatograms the bottom trace is an injec-tion of blank (Ringer solution). Samples kindly provided by Mrs. Gerdien Korte-Bouws, Department of Psychopharmacology, University of Utrecht, The Netherlands.

Linearity and repeatabilityUsing a dual channel configuration the acidic metabolites and the monoamines are measured (Fig. 4). They are loaded simultaneously on both HPLC’s in a single injection and ana-lyzed under completely different and optimized conditions.

The relative standard deviation (RSD) has been investigated for 8 injections of a mix of metabolites (100 nmol/L) and monoamines (10 nmol/L). The RSD in retention times is better than 0.5%. The RSD of peak areas and heights is better than 2% for the metabolites and better than 1.5 % for the mono-amines. Linearity shows a correlation coefficient better than 0.998 in the range of 2 to 100 nmol/L.

Table 1

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Conditions






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Nebulizer 1.6 Bar

Dry gas 8 L/min

Temperature 200 C


Hexapole 100 Vpp

Ion energy 5 eV

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®

Ordering numberALEXYS Neurotransmitter Analyzer for Monoaminessingle channel

180.0091U ALEXYS neurotransmitters, 1 ch

191.0035U AS 110 autosampler UHPLC cool 6p

180.0502 ALEXYS Monoamines SSC kit

dual channel

180.0092U ALEXYS neurotransmitters, 2 ch

191.0041U AS 110 autosampler UHPLC cool 10p

180.0502 ALEXYS Monoamines SSC kit (2x)

For research purpose only. The information shown in this communication is solely to demonstrate the applicability of the ALEXYS system. The actual performance may be aff ected by factors beyond Antec’s control. Specifi -cations mentioned in this application note are subject to change without further notice.

References1. Y. Ikarashi en Y. Maruyama, Determination of catechol-

amines, indoleamines, and related metabolites in rat brainwith LC with ECD, Biogenic amines 2 (1985) 101-110

2. J. P. Crombeen, J. C. Kraak en H. Poppe, Reversed-phase sys-tems for the analysis of catecholamines and related com-pounds by HPLC, J. Chromatogr., 167 (1978) 219-230

3. Nagao T, Tanimura T., Simultaneous determination of bio-genic amines, their precursors and metabolites in a singlebrain of the cricket using high-performance liquid chro-matography with amperometric detection, J Chromatogr.496(1) (1989) 39-53

4. Joseph MH, Kadam BV, Risby D., Simple high-performanceliquid chromatographic method for the concurrent deter-mination of the amine metabolites vanillylmandelic acid,3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleaceticacid, dihydroxy-phenylacetic acid and homovanillic acid inurine using electrochemical detection, J Chromatogr., 226(2) (1981) 361-368

5. Krstulovic AM, Dziedzic SW, Bertani-Dziedzic L, DiRico DE.,Plasma catecholamines in hypertension and pheochro-mocytoma determined using ion-pair RP chromatographywith amperometric detection: investigation of the separa-tion mechanism and clinical methodology. J Chromatogr.,217 (1981) 523-537

6. Katrien Thorré, Miloslav Pravda, Sophie Sarre, Guy Ebingerand Yvette Michotte, New antioxidant mixture for longterm stability of serotonin, dopamine and their metabo-lites in automated microbore liquid chromatography withdual electrochemical detection, Journal of Chromatogra-phy B: Biomedical Sciences and Applications, Volume 694,Issue 2, 4 (1997), 297-303

7. A.T. Nguyen, T. Aerts, D. Van Dam and P.P. De Deyn, Biogenic amines and their metabolites in mouse brain tissue: Devel-opment, optimization and validation of an analytical HPLCmethod, Journal of Chromatography B Volume 878, Issue29, 1 ( 2010) 3003-3014

8. Antec application note: 220_001 - Attomole detectionlimits

Robust Applications, Fluidly Running

Alexys Neurotransmitter Analyzer for Monoamines and Metabolites

IntroductionMicrodialysis of neurotransmitters in vivo has become an invaluable tool to study neurotransmis-sion in living brain. Cerebrospinal fluid of the brain is sampled trough a microdialysis device and analyzed by HPLC with electrochemical detection [1-7].

A neurotransmitter analyzer has been developed with features that meet the most demanding requirements. The required injection volume is small, as a smaller volume means a better time resolution in microdialysis.

The method has a low limit of detection, as some neurotransmitters have a concentration below 100 picomole/L. Separation and selectivity are optimized for multicomponent analysis, to get as much information as possible from a drop of dialysate. Where possible the analysis time has been shortened by any means such as by using UHPLC.

■ Fast separation on a sub-2μm UHPLC column

■ Wall-jet flow cell for best detection limit

■ Small injection volume, for better time resolution

■ Optimized separation for multi-component analysis

■ Dual channel option for parallel separations

®Application Note

Neuroscience

ALEXYS Analyzer for Highest Sensitivityin Neurotransmitter Analysis

Monoamines and Metabolites

Noradrenaline Dopamine Serotonin

5-hydroxyindole acetic acid (5-HIAA) 3,4-dihydroxyphenylacetic acid (DOPAC)homovanillic acid (HVA)

OPA derivatized amines and amino acids

GABA and GlutamateHistamine (LNAAs) 4-aminobutyrate (GABA)Glutamate (Glu)

LNAAs

Choline and Acetylcholine

Choline (Ch)Acetylcholine (ACh)

Markers for oxidative stress

3-nitro-L-Tyrosine 8-OH-DPAT

Glutathione and other thiols

ALEXYS Application Note # 213_028_04

Conclusion

The ALEXYS Neurotransmitter Ana-lyzer utilizes the extraordinary sep-aration power of sub-2 μm packed columns. Plate numbers, retention times and detection sensitivity have been pushed to their limits.

The application for Monoamines and their Metabolites in microdi-alysates is robust and suitable for routine based analyzes. Optimized method fi les are developed e.g. to prevent sample loss, to minimize re-tention times without compromises on the sensitivity and to inject sam-ples volumes of 2-10 uL. Typical detection limits of 50 pmol/L are feasible.

The ALEXYS Neurotransmitter Ana-lyzer can be extended with several options and kits for any combina-tion of other neurotransmitters, Acethylcholine and Choline as well as Amino Acids.

Analysis of microdialysis samplesThe matrix of microdialysate samples typically consist of the Ringer solution or artificial cerebrospinal fluid (aCSF) that is used for dialysis. The main constituent of these solutions is NaCl in a concentration of almost 0.15 mol/L.

Monoamines and some metabolites are not stabile in these solutions and break down rapidly. Adding a few microliters of concentrated preservative mix or acid to each collected frac-tion can prevent this. Care must be taken that the added pre-servative is not interfering with the chromatographic analysis later on.

Good results have been obtained using 1:4 addition of 0.1 mol/L acetic acid to samples in Ringer. Adding a high con-centration of perchloric acid (PCA) sometimes interferes with chromatography and causes deformation of peaks.

The results shown in this note are based on the analysis of standards in Ringer solution, acidified with acetic acid (final concentration 10 mmol/L HAc) unless mentioned otherwise. In case of UHPLC additional care must be taken that the sam-ple does not contain small particles. Centrifugation or filtra-tion is required in such case to avoid clogging of the column.To illustrate the applicability of the method a microdialysis fraction of basal level rat Prefrontal Cortex (PFC) has been an-alyzed (Fig. 5). Depending on the sample composition a little tuning might be required to get a bit more retention for NA. In case only DA and 5HT are of interest, the time of analysis can be decreased further.

The concentrations of monoamines in the microdialysis frac-tion are 0.24 (NA), 0.21 (DA), and 0.12 nmol/L (5HT). The con-centrations of metabolites are 2.6 (DOPAC), 36.1 (5-HIAA), and 7.8 nmol/L (HVA).

Under these conditions, the calculated detection limits in pmol/L are (brackets: amounts in fmol) for monoamines NA 80 (0.40), DA 70 (0.35), 5-HT 100 (0.50) and for the metabolites DOPAC 169 (0.85), 5-HIAA 69 (0.34), HVA 208 (1.04).

5

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Distributor:

Ihr Spezialist in der LC

ERC GmbH ☎ +49 89 66055696 Fax +49 89 60824826 [email protected] www.erc-hplc.de

www.myAntec.com

Documents

Oxidative Metabolism of Figure 10: Amodiaquine using the ... · The data for the MS Voltammogram were recorded using a scan mode with a potential range be-tween 0 and 1500mV, scanned