CE Pharm 2008, October 12-16, 2008, San Francisco, CA, USA

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1 Clinical Diagnostics by Capillary Electrophoresis: Determination of carbohydrate-deficient transferrin and lamotrigine in human serum under stringent quality control conditions CE Pharm 8, October 1-1, 8, San Francisco, CA, USA Wolfgang Thormann Department of Clinical Pharmacology and Visceral Research, University of Bern, Bern, Switzerland

2 Capillary electrophoresis in clinical/forensic diagnostics i) Proteins: serum proteins urine/csf proteins hemoglobins CDT lipoproteins ii) Molecular diagnostics (DNA): infectious diseases cancer genetics identity tests iii) Small molecules/ions: organic aciduarias thiols; nitrate, nitrite alcohol markers iv) Drugs/metabolites: therapeutic drugs drugs of abuse doping control Thormann et al., Electrophoresis (1999) 33 and (1) 1; Jabeen et al., Electrophoresis 7 () 13. Petersen and Mohammed, Clinical and Forensic Applications of Capillary Electrophoresis, Humana Press, 1.

3 CE instruments and reagents General instruments: P/ACE MDQ (Beckman Coulter) Agilent Technologies PrinCE, etc Reagents: CEofix (Analis) laboratory made buffers Multicapillary protein analyzers with reagent kits (walk away automation): Capillarys (Sebia); 8 capillaries Minicap (Sebia); capillaries Paragon CZE (Beckman); 7 capillaries

4 Two examples: CDT and lamotrigine levels in human serum PC + Capillary UV Detector - Buffer Samples Buffer Power Supply

5 Serum transferrin (Tf) - g/l, ~ % of total serum proteins isoforms with various Fe 3+ loads and N-glycan chains carbohydrate-deficient transferrin (CDT), marker for chronic alcohol abuse N N N N SA SA SA SA SA Fe 3+ Fe 3+ Fe 3+ SA SA SA Gal SA SA Fe 3+ C C C C SA = sialic acid, Gal = galactose % of total Tf (healthy person) pi *) 8-sialo-Tf ND 5. 7-sialo-Tf < sialo-tf sialo-Tf sialo-tf sialo-Tf sialo-tf < sialo-Tf ND 5.8 -sialo-tf ND 5.9 *) complete iron saturation of C1-Tf adapted from Arndt, Clin. Chem. 7 (1) 13. CDT

6 High-resolution CDT analysis based on CEofix CDT reagents of Analis Sample preparation: serum + Fe 3+ solution ( μl each) P/ACE MDQ (Beckman Coulter) Capillary: 5 μm ID x cm total length Conditioner:. M NaOH Initiator: polycation in Tris/phosphate, ph. Buffer: polyanion in Tris/borate, ph 8.5 Injection:.5 psi x 1. s (vacuum) Separation: kv, 3 C Detection: nm Sample throughput:.7/h 5 cm capillary/8 kv/3 C: ABSORBANCE (mau) CURRENT (μa) VOLTAGE (kv) cations Tf immunosubtraction electroosmosis TIME (min) Tf-pattern γ fraction voltage current 5 samples/h - lower resolution Joneli et al., J. Chromatogr. A 113 () 7.

7 ABSORBANCE (mau) High-resolution CEofix electropherograms : asialo-tf : disialo-tf 3: trisialo-tf : tetrasialo-tf 5: pentasialo-tf : hexasialo-tf TIME (min) 5 5 Normal serum: CDT: 1.7 % di/trisialo-tf Rs = 1.9 CZE data: : ND : 1.7 % 3: 7.81 % : 7.51 % 5: 15.5 % : 3.1 % Alcohol abuser: CDT: 1.77 % di/trisialo-tf Rs = 1. CZE data: : 1.77 % : 9. % 3: 3.88 % :.3 % 5: % : 3.5 % Interday precision for CDT: RSD levels of %

8 Data comparison: high-resolution CEofix vs. Capillarys of Sebia 1 Rs = 1.7 Capillarys method (Sebia) 8 y = 1.x -.5 n = r = High-resolution CEofix method on MDQ Marti et al., J. Sep. Sci. 31 (8) 379. Rs = 1.3 method High-resolution CEofix: CDT (%) Capillarys: CDT (%) mean median 1..8 range cut-off 1.7 % 1.3 %

9 Internal quality control C U R R E N T Y E A R Year 5 7 CDT level: 1. % ( ) mean: 1. % RSD: 5.7 % n Mean (%) RSD (%) ABSORBANCE (mau) ABSORBANCE (mau) 1 1 Commercial controls Recipe high CZE CDT: 3.99 % (:.53 %; : 3. %) BioRad Cal CZE CDT: 5.7 % TIME (min)

10 External quality control GTFC proficiency test, markers of alcoholism in serum: AMF 3/7, sample B: positive ABSORBANCE (mau) 3 13 AMF 3/7 B CZE CDT: 3.73 % (:. %; : 3.7 %) TIME (min) 5 results of 57 participants: Assay CE Sebia HPLC IA n Mean (%) RSD (%)

11 Pros and cons of CDT analysis by CE Pros: Sample preparation Automation Imprecision (RSD < 7 %) High resolution / high throughput Abnormal Tf patterns Cons: Unselective detection ( nm) Occasional interferences and/or insufficient Tf ABSORBANCE (mau) Tf-CD variant D 3D D γ D+3C C 5C C C C 5D D TIME (min) J. Chromatogr. A 113 () 7. ABSORBANCE (mau) Before and after IgY Tf purification EO γ TIME (min) 3 3 β 5 5 J. Chromatogr. A 1 (8) 33.

12 TDM of antiepileptic drug lamotrigine serum/plasma: CZE assay with optical detection therapeutic range: 8 - μm (ca. - μg/ml) PP: 5 μl sample + 1 μl acetonitrile containing 3 μg/ml tyramine (IS) acidification with 1 μl.9 M acetic acid, 5 psi x s injection CZE: 13 mm sodium acetate/acetic acid (ph.8 or.5) BioRad 3: 5 μm ID cap. of 3 cm length, 1 kv ( μa), C, 1 nm detection limit: μm; RSD < % (interday); throughput: 7 s/h J. Chromatogr. B 83 (199) 119 J. Chromatogr. A 979 () 353 C A LI B R A TI O N with bovine plasma ABSORBANCE [mau] AREA RATIO CONC. [μm]. IST IST IST LAMO IST LAMO IST LAMO LAMO LAMO TIME [min] * * * * ABSORBANCE [mau] 8 3TD IST LAMO IST LAMO patient IST LAMO control TIME [min] * * * S A M PL E S

13 QC of lamotrigine TDM External quality control data Internal quality control data Drug level: 9.75 μm Year n Mean (μm) RSD (%) J. Chromatogr. A 979 () 353

14 Conclusions CE is highly suited for clinical and forensic diagnostics: resolution, precision, robustness, sample preparation Commercial instruments and reagents permit routine use of CE Further promotion of CE technology: new approaches, chip-based assays Acknowledgements C. Lanz, F. Tagliaro, F. Bortolotti, M. Kuhn, U. Marti, V. Deiss, J.-B. Falmagne, F. de l Escaille, J. Joneli, U. Wanzenried, J. Schiess R. Theurillat, M. Kuhn, J. Joneli, U. Wanzenried, J. Schiess Swiss National Science Foundation / Liver Foundation Bern Analis, Suarlée, Belgium

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