Drug analysis by capillary electrophoresis and laser-induced fluorescence

Electrophoresis 1998, 19, 2777-2790 Drug analysis by CE-LIF 2777

Review

Franqois Couderc' Elizabeth CaussC2 Christophe Ba~le'>~

'Universitt! Paul Sabatier, Laboratoire de Biologie MoICculaire Eucaryotes, Toulouse Cedex, France 2CHU Rangueil, Biochimie, Toulouse, France 3Zeta Technology, Ramonville St. Agne, France

Contents

1 2 2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.3 2.4 3 3.1

3.1.1 3.1.1.1 3.1.1.1.1 3.1.1.1.2 3.1.1.2 3.1.2 3.1.2.1 3.1.2.1.1

Drug analysis by capillary electrophoresis and laser-induced fluorescence

This review briefly presents the different laser-induced fluorescence detectors, outlines the different dyes used to derivatize molecules which are used with capillary electrophoresisflaser-induced fluorescence (CE-LIF), and provides an overview and current status of CE-LIF in drug analysis.

Introduction . . . . . . . . . . . . . . . . . . . . 2777 Laser-induced fluorescence detector . . . 2777 Principles . . . . . . . . . . . . . . . . . . . . . . 2777 Instrumentation . . . . . . . . . . . . . . . . . . 2778 Beckman LIF detector . . . . . . . . . . . . . 2778 Zeta LIF detector . . . . . . . . . . . . . . . . 2778 Perkin Elmer LIF detector . . . . . . . . . . 2778 Bio-Rad detector . . . . . . . . . . . . . . . . . 2778 Derivatization and useful laser . . . . . . . 2778 Sensitivity performance . . . . . . . . . . . . 2778 CE-LIF and drug analysis . . . . . . . . . . 2780 Direct analysis of fluorescent drugs by

Visible wavelength excitation . . . . . . . . 2780 Anticancer drugs . . . . . . . . . . . . . . . . . 2780 Free anthracyclines and metabolites . . . 2780 Doxorubicine immunoconjugate . . . . . . 2781 Antineoplastic drugs . . . . . . . . . . . . . . 278 1 UV wavelength excitation . . . . . . . . . . 278 1 Use of a 325 nm He-Cd laser . . . . . . . . 2781 Anticancer drugs . . . . . . . . . . . . . . . . . 278 1

3.1.2.1.1.1 Methothrexate . . . . . . . . . . . . . . . . . . . 278 1 3.1.2.1.1.2 Cis-platin and carboplatin . . . . . . . . . . 278 1 3.1.2.1.1.3 6-Mercaptopurine . . . . . . . . . . . . . . . . 2782 3.1.2.1.2 Vitamins . . . . . . . . . . . . . . . . . . . . . . . 2782 3.1.2.1.3 Hypnotic drugs . . . . . . . . . . . . . . . . . . 2782 3.1.2.1.4 Anti-inflammatory drug . . . . . . . . . . . . 2783 3.1.2.2 Other near-UV wavelengths . . . . . . . . . 2783 3.1.2.3 Low-UV laser wavelength . . . . . . . . . . 2783 3.1.2.3.1 Alzheimer disease drug . . . . . . . . . . . . 2783 3.1.2.3.2 Quality control of biopharmaceuticals . . 2784 3.1.2.3.3 Insulin . . . . . . . . . . . . . . . . . . . . . . . . 2784 3.1.2.3.4 Impurities of illicit heroin . . . . . . . . . . 2784 3.2 Immunoassay using CE-LIF . . . . . . . . . 2784 3.2.1 Competitive immunoassay . . . . . . . . . . 2784

CE-LIF . . . . . . . . . . . . . . . . . . . . . . . . 2780

Correspondence: Dr. F. Couderc, Universitk Paul Sabatier, Laboratoire de Biologie MoMculaire Eucaryotes, 118 Route de Narbone, F-31062 Toulouse Cedex, France (Tel: +33-5-6117-5499; Fax: +33-5-6133-5886; E-mail: [email protected])

Abbreviations: CZE, capillary zone electrophoresis; IST, salicylate; LOD, limit of detection; mAb, monoclonal antibody; NAP, naproxen

Keywords: Capillary electrophoresis / Drug / Laser-induced fluorescence I Review

3.2.2 Noncompetitive assay . . . . . . . . . . . . . 3.2.3 3.2.3.1 3.2.3.2 3.2.3.3 3.2.3.4 3.2.3.5

3.2.3.6 4 5

2785 Applications . . . . . . . . . . . . . . . . . . . . 2785 Insulin quantitation . . . . . . . . . . . . . . . 2785 Human growth hormone . . . . . . . . . . . 2785 Morphine, phenyclidine . . . . . . . . . . . . 2786

Theophylline, ethosuximide, paracetamol, salicylate, and quinine . . . . . . . . . . . . . 2787 Vitamin A . . . . . . . . . . . . . . . . . . . . . 2787 Conclusion . . . . . . . . . . . . . . . . . . . . . 2788 References . . . . . . . . . . . . . . . . . . . . . 2788

Digoxin . . . . . . . . . . . . . . . . . . . . . . . 2786

1 Introduction During the past 10 years, instrumentation for electrokinetic separations in fused-silica capillaries, known as capillary electrophoresis (CE) has become available and its use in clinical and pharmaceutical laboratories has begun to be explored intensively [l-51. Most of the studies concerning drugs have been completed with ultraviolethisible (UV- Vis) direct or indirect absorption. Due to the need in sensitivity, drug monitoring in body fluids necessitates the use of laser-induced fluorescence (LIF) as a detector. Although there are nearly a dozen companies manufactur- ing electrokinetic capillary instrumentation [6] , only four are able to sell LIF detectors, whose price is twice that of UV-Vis diode array detectors. Moreover, if laser-induced fluorecence detection is a sensitive method, the lack of natural native fluorescence of most molecules implies a need to tag them with fluorescent dyes. These limitations explain the small amount of literature in drug analysis using CE-LIF. The purpose of this review is to present the different optical arrangements of laser-induced fluorescence detectors and to provide a brief overview and current status of CE-LIF in drug analysis.

2 Laser-induced fluorescence detector

2.1 Principles Conventional fluorescence has been successfully applied in conventional high performance liquid chromato raph yielding detection limits ranging from lo-" to 10- moll L [7]. The sensitivity can be enhanced considerably by increasing the power of the monochromatic light source, since the fluorescence signal observed is proportional to the

l i y

0 WILEY-VCH Verlag GmbH, 69451 Weinheim, 1998 0173-0835/98/1617-2777 $17.50+.50/0

2778 F. Couderc, E. Causse and C . Bayle Electrophoresis 1998, 19, 2111-2190

applied excitation power. For this purpose laser systems are useful because the light source provides extremely intense collimated light beams of highly monochromatic radiations with output powers up to several watts. In laser-induced fluorescence detection systems, excitation power may be increased four to six orders of magnitude compared to conventional fluorescence detection, resulting in a much better signal-to-noise ratio, although background noise signals may be elevated as well as a result of intensified scattering of excitation radiation. Various laboratories have developed different LIF detectors in the past 15 years. They were first used with HPLC and transposed for CE. Several authors have described the different arrangements used for CE analysis [8]; for a detailed review on LIF detector optical arrangements see Nouadje et al. [9]. Only four companies sell the LIF detector: Beckman Instruments (Fullertown CA, USA) on the PACE system, Zeta Technology (Modular instrument), (Ramonville, France), Perkin Elmer (Norwalk, CT, USA) and Bio-Rad (Hercules, CA, USA) on the CE instrument [6]. The present review addresses the optical arrangements of the four commercially available instruments.

2.2 Instrumentation

2.2.1 Beckman LIF detector The Beckman LIF detector is dedicated to the CE PACE instrument. An optical fiber ended by a small on-end fiber lens delivers the laser beam to the capillary and, to improve sensitivity, a parabolic mirror reflects the fluorescence to the photomultiplier tube (PMT). Not all of the laser power is concentrated in the inner diameter of the capillary. An appropriate filter kit minimizes the background noise. No adjustment of the laser beam in front of the capillary is allowed. This detector is used with several laser wavelengths, from 647 to 325 nm.

2.2.2 Zeta LIF detector

This detector is based on the colinear arrangement of an epi- illumination microscope. The laser beam is deviated by a dichroic mirror and focused on the inner diameter of the capillary using a long focus distance lens and a small sapphire ball lens. It illuminates the inner diameter of the capillary and improves the illuminated volume. Moreover, the very high numerical aperture of the 2 mm ball lens provides a large amount of fluorescence which is partic- ularly useful to increase the sensitivity of the detection [lo]. The colinear arrangement is also useful to minimize the Raman background noise. The position of the laser beam into the capillary can be easily adjusted by the user to improve the sensitivity. Most of the laser wavelengths have been used with this detector (647-325 nm).

2.2.3 Perkin Elmer LIF detector

This detector is dedicated to a capillary DNA analyzer or sequencer (ABI PRISM, Perkin-Elmer, Norwalk, CT, USA). It has a colinear arrangement. The 488 and 514 nm wavelengths of an argon laser are colimated via a ball lens on the capillary. A dichroic mirror and a filter kit make it possible to obtain fluorescence without reflections of the

laser wavelengths. A monochromator and a 64 X 152 charge-coupled device (CCD) camera, cooled by two Peltier devices, record the fluorescence spectra along the analysis. When using different fluorotides for DNA sequencing, the emission spectra are used to identify the different tags. To our knowledge, there is no limit of detection (LOD) in the literature.

2.2.4 Bio-Rad detector

This two-excitation wavelength LIF detector, mainly used for nucleic acids analysis, uses two laser tubes : an argon- ion laser (488 nm) and an He-Ne laser (647 nm). The laser beams are focused on two optical fibers which illuminate the capillary. The fluorescence is collected orthogonally and the fluorescence induced by the different lasers can be recorded.

2.3 Derivatization and useful laser

The main limitation of capillary electrophoresis is the unfavorable concentration detection limits frequently obtained. Although the mass detection limits reported can be withn the pmol to fmol range, the concentration detection limits are normally significantly higher because the injection and, consequently, the detection volumes are in the nL range. This means that, in order to obtain the same sensitivity as in liquid chromatography (LC), special detection techniques, such as LIF, must be used to detect the analytes better. It is often useful to derivatize the molecules. Pre- and postseparation derivatizations have been described previously in the literature for different functional groups [ll]. Table 1 summarizes the different tags used. One of the major problems of molecule labeling is the reaction yield of the functional group. If reactions are performed with concentrations around lo4 M, there are no labeling problems. If derivatization is performed with a concentration less than lop7 M, some authors found that derivatization (e.g. for amino acids with FITC or dichlor- otriazinylaminofluoresceine (DTAF)) was then not quantitative [23]. On the other hand, Robert et al. [33] demonstrated that they were able to derivatize amino acids and catechol amines at concentrations of lo-'' M in 1 pL of rat microdialysis using naphthalene dicardoxalyldehyde (NDA). In conclusion, functional group labeling is a problem that may be difficult to solve. Moreover, fluorescence yield of the fluorophore can be reduced after derivatization.

2.4 Sensitivity performance

The LIF pre- or postcolumn derivatization is often carried out to detect various biological molecules. Unfortunately, these methods can result in a number of problems when detecting proteins and peptides. Because derivatization can cause multiple labeling of analytes with more than one derivatization site, as is the case with most proteins and peptides, native fluorescence is then advantageous. A varying number of derivatized sites will cause different electrophoretic mobilities for the same analyte, resulting in multiple peaks. Using native fluorescence alleviates this problem of multiple labeling. With an excitation at 275 nm,

Electrophoresis 1998, 19, 2777-2790

Table 1. Fluorescent markers used for different chemical functions with CE-LIF“’

Drug analysis by CE-LIF 2779

~ ~

Function Reagent Compound Limit of Excitation References detection wavelength (LOW laser tube

Amines AEOC Benzoin

CBQCA

CTSP DCC

DCCS DNSH DTAF FITC

Fluorescamine

FMOC NBD-F

NDA

OPA

TRIC

Amino acids Arginine-containing peptides Amino acids Peptides Amino sugars Amino acids Amino acids, small peptides Amino acids Toxins Amino acids Amino acids Peptides Proteins Peptides Amino acids Toxines Amino acids Amino acids Dipeptides Amino acids Catechol amines Jeffamine polymers Toxins Amino acids Amino acids

Keto or aldehyde of carbohydrates AC SugarsPoly saccharides AHNS Monosaccharides 3-ANDA Oligosaccharides 5-ANSA Monosaccharides ANTS Oligosaccharides AP Mono/oligosaccharides APTS Mono/oligosaccharides AQ Charged

CBQCA Mono/oligosaccharides TRSE Monosaccharides

MPD Peptides

AAF Phenoxy acid

7-ANDA Phenoxy acid

PDAM Dicarboxylic acid MF Short and long

oligosaccharide

Carbonyl

Carboxyl

herbicides

herbicides

fatty acids Sulfiydryl

Cys 5.3aIA Cys 5.4aIA MBrB Glutathione NDA Glutathione OPA

0.11150 amol 351 nm, Ar+ 270 amol 325 nm, He-Cd

9 zmol 488 nm, Ar+ 4-70 amol 488 nm, Ar+ 240 am01 488 nm, Ar+ 1-7 am01 663 nm, Diode 0.1 amol Red diode

100 amol 415 nm, Diode 325 nm, He-Cd

amol 488 nm, Ar+ 9zmol-lam01 488 nm, Ar+

488 nm, Ar’ 280 nm 365 nm, Ai’

24 fmol 390 nm

0.8 fmol, 0.2 amol 351 nm, Ar+ 488 nm, Ar+

70 amol 488 nm, Ar+ 12 am01 442 nm, He-Cd 0.5 amol 442 nm, He-Cd

442 nm, He-Cd 0.1 amol 325 nm, He-Cd 80 am01 325 nm, He-Cd 1 zmol 543 nm. He-Ne

488 nm, Ar’ 325 nm, He-Cd 325 nm, He-Cd 325 nm, He-Cd 325 nm, He-Cd 325 nm, He-Cd

325 nm, He-Cd 2 pmol 488nm, Ar+

0.5 amol 488 nm, Ar+ 17 zmol 514 nm

390 amol 488 nm, Arf [15]

3 amol 488 nm, Arf [481

am01 325 nm, He-Cd [49]

488 nm, Ar+ [50] 488 nm, Ar+ [51]

670 nm, diode [52] 635 nm, diode [52]

amol 355 nm, Ar+ [53] am01 458 nm, Ar+ [54]

325 nm, He-Cd [55]

a) AAF, 5-(aminoacetamido)fluorescein; AC, 2-aminoacridone; ACE, affinity capillary electrophoresis; AEOC, 2-(9-anthryl)ethylchloroformate; AHNS, 4-amino-5-hydroxynaphthalene-2,7-sulfonic acid; 3-ANDA, 3-aminonaphthalene-2,7-disulfonic acid; 7-ANDA, 7-aminonaphthalene-1,3-disulfonic acid; 5-ANSA, 2- aminonaphthalene-1-sulfonic acid; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; AP, 2-aminopyridine; APTS, 9-aminopyrene-l,4,6-trisulfonate; AQ, 6-aminoquinoline; CBQCA, 3-(4-carboxybenzoyl)-2-quinoline carboxaldehyde; CTSP, 9-cyano-N,N,N’-triethyl-N’-(5-succinimidy~oxycarbonylpentyl)pyronin; DCC, dicarbo- cyanine reagent; DCCS, 7-(diethylamino)coumarin-3-carboxylic acid succinimidyl ester; DNSH, 5-(dimethyl- amino)naphthalene-1-sulfonehydrazine; DTAF, dichlorotriazinylaminofluorescein; FMOC, 9-fluorenyl-methyl chloroformate; HGH, human growth hormone; MBrB, monobromobimane; MF, 5-bromomethym-fluorescein; MPD, 4-methoxy-l,2-phenylenediamine; NBD-F, 4-fluoro-7-nitro-2,1,3-benzoxadiazole; NDA, naphthalene dicardoxaldehyde; OPA, o-phthaldialdehyde; PDAM, 1-pyrenyldiazomethane; PMT, photo multiplier tube; TFUC, tetramethylrhodamine isothiocyanate; TRSE, 5-carboxytetramethylrhodamine

2780 F. Couderc, E. CaussC and C. Bayle Electrophoresis 1998, 19, 2777-2790

therefore, tyrosine and tryptophane can be detected without any need of chemical derivatization. With a KrF laser other workers [56, 571 used the 248 nm wavelength to im rove the detection of tryptophane (7 X M, 7 X 10- M). Polycyclic aromatic hydrocarbons were also detected in such a way [58, 591. The limitation in this field is the much higher cost of a UV laser compared to a conventional visible laser. The powerful diode lasers and the improvement of frequency doubling or tripling will result in inexpensive UV lasers in the next few months and, consequently, in the increased use of LIF detection.

Po

3 CE-LIF' and drug analysis

The two main strategies to detect and quantitate drugs with CE-LIF are direct analysis of fluorescent drugs or immunoassays using labeled antibodies. Of the large amount of work published on labeling reactions, only few relate to the

use of CE-LIF with chemically labeled drugs. Actually, most of the work on drugs using CE was done with CE and UV detection [3, 51.

3.1 Direct analysis of fluorescent drugs by CE-LIF

3.1.1 Visible wavelength excitation

3.1.1.1 Anticancer drugs

3.1.1.1.1 Free anthracyclines and metabolites

One of the first studies on drugs using an argon-ion laser for CE-LIF analysis was performed on anthracyclines. Rhein- houd et al. [60] analyzed anthracycline in plasma using CE- LIF with the 488 nm wavelength of an argon-ion laser. Doxorubicin, daunorubicin and epirubicin fluoresce naturally at 488 nm. The sensitivity obtained is 0.5-1 ng/mL.

1 ) K O

I I

Figure 1. (A) Distribution of possible conjugated species from monoclonal antibody (mAb) BR96-doxorubicine immunoconjugate upon denaturation. (B) SDS-capillary gel electrophoretic separation of mAb BR96 and mAb BR96-doxorubicin immunoconjugate with laser-induced fluorescence detection. (a) mAb BR96-Dox immunoconjugate under denaturation conditions; (b) mAb BR96-Dox immunoconjugate under denaturation and reduced conditions; (c) mAb BR96 under denaturation and reduced conditions [64].

on ion mn X O

nme (rntn)

Eiectruphuresis 1998, 19, 2111-2790 Drug analysis by CE-LIF 2781

The author presents an electropherogram of these anthracyclines from an extract of plasma sample taken 120 min after administration to a patient treated intravenously with epirubicin. Daunorubicin was used as an internal standard.

Hempel G. et al. [61] looked for doxorubicinol, a metabolite of doxorubicine. Doxorubicinol was monitored over 8 h in patients' plasma treated with doxorubicin. In the same manner the authors [62] proposed the quantitation of idarubicin and its metabolite idarubicinol in plasma. The small sample volume of 100 pL is of particular advantage for studies in pediatric oncology. The reproducibility of the method has been shown to be sufficient for drug monitoring or pharmacokinetic studies. The limit of quantification for idarubicin in plasma is 0.5 ng/mL [62].

3.1.1.1.2 Doxorubicine immunoconjugate

The potential usefulness of monoclonal antibody conjugated with antitumor drug provides a highly selective vehicle for drug delivery to the tumor site [63]. Recognition of the target cell and the consequent increase in the local concentration of drug allows for site-directed killing of the cell. Monoclonal antibodies possess many potential sites for drug attachment. In an ideal situation, the immunocon-

Syringe Pump #2

A [w Transfer Ljne A

Syringe Pump X I Interface

/

'OW0 r-----l h l i 8COO

A

Time (seconds)

Figure 2. (A) Scheme of the on-line microdialysis/capillary electrophoresis system. (B) Typical electropherograms of blood dialysates using the on- line microdialysidCE system. Samples: (A) to a blank prior to dosing: (B) 10 min after a 4 mgkg i.p. dose of SR 4233. Peak 1, SR 4233: 2, SR 4317 (main metabolite of SR 4233) [65].

jugate would carry the maximum number of drugs per molecule of antibody while retaining immunoreactivity and specificity. Liu et al. [64] presented an analysis of monoclonal antibody chimeric BR96-doxorubicine immunoconjugate by SDS-capillary gel electrophoresis and laser- induced fluorescence. The authors used these separation and detection means for the analysis of monoclonal antibody chimeric BR 96 and the corresponding immunoconjugate prepared between BR 96 and the anticancer drug doxorubicin. These molecules were studied in their native, denatured and reduced status. Six peaks were identified following separation of the denatured bioconjugated molecule. As expected, they corresponded to all possible conjugated species. Analysis of the resulting fingerprint maps indicated that the light, heavy, and light-heavy chain conjugates were the predominant species. Figure 1 presents the possible conjugated species from the immunoconjugated species upon denaturation and the separation of the different denaturated species [64].

3.1.1.2 Antineoplastic drug

In a study on coupled microdialysis CE, Hogan et al. [65] monitored the antineoplastic drug SR 4233 in free-moving rats using the 442 nm He-Cd laser. Figure 2A shows the design of the on-line microdialysisicapillary electrophoresis system. Typical electropherograms of blood dialysates showed SR 4233 baseline-resolved from its main metabolite within a 60 s run time (Fig. 2B). The CE-LIF procedure permitted pharmacokinetic studies.

3.1.2 UV wavelength excitation

3.1.2.1 Use of 325 nm He-Cd laser

3.1.2.1.1 Anticancer drugs

3.1.2.1.1.1 Methothrexate This was the first measurement using CE-LIF and a UV wavelength. Roach et aE. [66] demonstrated that they could quantitate methotrexate and 7-hydroxy-methotrexate in clinical sam les. The detection limits were 5 X lo-'' M and 2 X 10- M, respectively. The extraction efficiency for the drug and its metabolite from serum was around 85% using Sep-Pak cartridge. Methotrexate and its metabolite were oxidized with potassium permanganate to obtain detectable fluorescent molecules.

g

3.1.2.1.1.2 Cis-platin and carboplatin

The micellar electrokinetic capillary chromatographic separation of a dansylated mixture of normal nucleotides and platinated cross-link adducts of d(pGpG) and d(pApG) was realized by Sharma et al. [67]. CELIF was able to detect 1 adduct/104 normal nucleotides/mg DNA by fluorescence post-labeling assay. The enrichment of the adduct, prior to dansylation, enhanced the detection limit to 1 adduct/107 normal nucleotides/mg DNA. Calf thymus DNA reacted in v i m with cis-platin and carboplatin with total input drug/nucleotide ratios of 0.05 and 0.5, respectively. For 2 h 2780 human ovarian carcinoma cultured cells were exposed to 25 mM cis-platin. The cells were incubated

2782 F. Couderc, E. Causd and C. Bayle Electrophoresis 1998, 19, 2111-2790

I I I 8 16

I 8

c>

-

I 12

'1 I 16

I I I 0 12 16

Time (min 1

Figure 3. CELIF analysis of (a) dansylated 1,2 intra-strand cross-link of d(pGpG) and d(pApG) and post-labeled DNA digests from 5 ng; (b) cis- platin exposed A2780 human ovarian carcinoma cells and (c) mock-treated A2780 cells [67].

with drug-free medium for 3 h before harvesting. The identification of the cross-link adducts in the modified DNA was confirmed by cochromatography with the authentic markers. The same 1 ,Zintra-strand cross-link adducts were induced by both cis-platin and its second generation drug carboplatin. Figure 3 [67] shows the CE-LIF analysis of a dansylated 1,Zintra-strand cross-link of d(pGpG) and d(pApG) and postlabeled DNA digest of cis-platin-exposed carcinoma cells.

3.1.2.1.1.3 6-Mercaptopurine

6-Mercaptopurine and its metabolite were separated and detected with CE-LIF. The 6-thioguanine nucleotides were oxidized in order to produce fluorescent species with potassium permanganate. The limits of detection are around 2 X lo-'' M [68]. The method of extraction proposed by the authors is simpler than that of the classical studies involving mercury cellulose resins or phenyl mercury adduct formation.

3.1.2.1.2 Vitamins

Burton et al. [69] proposed the analysis of B6 vitamins using MEKCLIF. In capillaries of 25-75 pm ID they separated vitamin B6 and its metabolite. An efficiency as high as 60000 theoretical platedm is obtained. Pyridoxic acid, a metabolite of B6, was separated and quantitated in human urine. Limits of detection were less than a picogram injected and plates were achieved. This work was also done by Kurosu et al. [70] using conventional fluorescence to analyze the vitamin B6 group.

3.1.2.1.3 Hypnotic drugs

Zolpidem and zopiclone were analyzed using CE-LIF. Zolpidem, which is an imidazopyridine molecule, has been reported to be analyzed with its main metabolite without extraction from urine. A 10 min separation provides an LOD of 2 ng/mL [69]. A method has been developed also for the stereoselective determination of zopliclone (cyclo- pyrrolone drug ) and its main metabolites in urine. In this case they are extracted from urine or saliva. The S-(+)- enantiomer and its metabolites were always extracted in higher amounts than R-(-)-enantiomen. Figure 4 [7 11

l b

I

, I , I ~, , , 1 , .""" 0

8.00 in.00 u.00 14.00 [mnl

Figure 4. Electropherograrns of (a) human urine 6 h after oral administration of zopiclone and (b) blank urine [72].


presents the separation graph of these compounds from a human urine extract [72]. These drugs can be easily quantitated.

3.1.2.1.4 Anti-inflammatory drug

Soini et al. [73] described a method to determine naxopren (NAP) in serum. Sample preparation includes a simple liquid-liquid extraction of 0.25 mL serum aliquots. After solvent evaporation, samples were dissolved in methanol and analyzed using CE-LIF. The method provides interfer- ence-free, linear and highly reproducible results. A quantitation limit of 3 fmol was achieved. Using warfarin as internal standard improved the analytical precision substantially. The CE-LIF method is 30 times more sensitive than CE-UV detection. Using micellar electrokinetic capillary electrophoresis (MEKC), Albrecht et al. [74] reported the separation of NAP from naproxen covalently coupled to human serum albumin (NAP-HSA) or to mannosylated serum albumin (NAP-HSA-MAN) and the metabolite naproxen-lysine (NAP-LYS). An assay for selective analysis of the different forms of NAP by direct plasma injection was canied out with salicylate (IST) as internal standard and detection by laser-induced fluorescence (Fig. 5). MEKC offers the advantage that free and

NAP-HSA-MAN

$ 9.5 N

U rr -0.5 ry -0.5

19.5

14.5 E

c? u) 9.5

c co co

(u 0 3 u. of

I 4.5

I ST

NAP

NAP-HSA-MAN

-0.5 0 2 4 6 a

Time (min)

Figure 5. Separation of different naproxen-protein conjugates WAF') after direct injection of rat plasma supplemented with naproxen-human serum albumin (NAP-HSA at 50 m&), naproxen-mannosylated human serum albumin (NAP-HSA-MAN at 125 m&) and salicylate (IST, at 50 mg/L). Data obtained via injection of calibrators (without plasma) are depicted in the insets.

covalently bound NAP can be differentiated in one run and can be accurately monitored in microliter quantities of plasma. In the same way, NAP was identified in liver and kidney tissues [75]. Detection limits were 140 X lo-' m o m of NAP and 7 X lo-' m o m of NAP-HSA [74].

3.1.2.2 Other near-W wavelengths

Pulsed nitrogen laser was used for banned substances in sport. This type of laser provides enormous energy to be concentrated into a small sample volume, for example a modest 2.5 mJ, 10 ns pulse focused to a 75 pm diameter spot results in an irradiance of 5.6 X 10' Wcm-'. This great photon flux is capable of detecting a single molecule. Pulsed lasers require gated detection devices for signal recovery and accurate timing for synchronization of the excitation-detection cycles. The triamterene LOD was reported to be 3.1 mol, acebutolol 8.8 X mol, bendroflumethiazide 1.8 X mol. Triamterene was identified in urine [76].

3.1.2.3 Low-W laser wavelength

Only few studies have been performed using low-UV laser wavelength for the CE-LIF study of drugs, due to the cost of such a laser tube. Moreover, these studies were performed on homemade laser detectors. We will summarize the different applications.

3.1.2.3.1 Alzheimer disease drug

NXX-066 (Fig. 6) [77], which is naturally fluorescent at La, = 21 1 nm, has been studied using LIF detection with a frequency-doubled argon-ion laser delivering a 244 nm

Figure 6. CELIF, analysis of NXX-066 at 1 X candidate drug intended for treatment of Alzheimer disease) [77].

M (NXX-066 is a

2784 F. Couderc, E. Causst and C. Bayle Elecirophoresis 1998, 19, 2111-2190

0 2 4 a a 10 12

Time (mln)

Figure 7. Electropherogram of purified anti-TAC (28 mg/mL or 180 p~ WI.

wavelength (5 mW). An LOD of 5.1 X M was achieved and the separation was performed using the isotachophore- sis principle. The improvement in sensitivity of CE-LIF was 55 times higher than CE-UV.

3.1.2.3.2 Quality control of biopharmaceuticals

The use of living organisms for the manufacture of pharmaceuticals by recombinant DNA technologies presents a unique approach and promises to transform numerous aspects of human health care. Although many analytical techniques developed in the biological sciences have been applied to quality control of biopharmaceuticals, the necessity of accuracy, sensitivity, resolution and speed appears to be found in using CE-LIF. All the biopharmaceuticals studied by Lee et al. [78] can be detected at subnanomolar level with linear dynamic ranges of at least three orders of magnitude. CE-LIF, using the 275.4 nm line of an argon-ion laser, can determine impurities in 'purified' biopharmaceuticals present in amounts less than 0.01 % of the major component. Figure 7 [78] shows the analysis of the purified anti-T activating antigen (TAC) protein.

3.1.2.3.3 Insulin

Insulin, intensively studied with imunoassays, was also analyzed using CE-LIF and direct fluorescence detection

I A

[ I i o io io i o SO do

Time (min)

with the 275.4 nm argon-ion laser [79, 801. In these studies, a nonbonded poly(ethy1eneoxide)-coated capillary was used to achieve the separation. An insulin concentration as low as 73 am01 can then be detected.

3.1.2.3.4 Impurities of illicit heroin

Lurie et al. [81] described the separation and the detection of acidic and neutral impurities in illicit heroin. Separations were achieved using charged cyclodextrine-modified MEKC. Phenanthrene-like heroin impurities exhibit high native fluorescence under krypton-fluoride laser excitation (248 nm). The limit of LIF detection for one of these solutes (acetylthebaol) was 1.8 ng/mL, 500 times more sensitive than UV. Figure 8 [81] is a comparison of UV detection at 260 nm and LIF detection of a refined Southwest Asian heroin. This methodology is applicable to analysis of both crude and refined heroin.

3.2 Immunoassay using CE-LIF

The study of antibody-antigen reactions by CE was demonstrated by Nielsen et al. [82]. Immunoassays have become the method of choice for a wide variety of clinical pharmaceutical and chemical analyses owing to their ability to quantitatively measure small amounts of analyte (antigen) in a complex mixture whose constituents are often at much higher concentrations [83,84]. The high selectivity of such an assay is achieved by using antibody reagents possessing high discriminatory power and high binding affinity for the analyte of interest even in complicated matrices. The high sensitivity of immunoassay analysis is due to the use of specific labels covalently linked to one of the reactants, antibody or antigens, for detection. The CE immunoassays which were reported are either in the direct or the competitive format. These assays are all solution- phase assays in which all assay components are present in a dissolved form during incubation. To date, only preliminary feasibility studies have been presented, with no single CE assay being completed so that it fulfills the rigorous analytical standards set for immunoassays.

3.2.1 Competitive immunoassay

Figure 9 [85] illustrates the basics of the assay procedure. First, a predetermined amount of labeled antigen is added to

0 10 20 30 40 50 60 Time (min)

Figure 8. Comparison of (A) UV detection at 260 nm and (B) LIF detection with a krypton-fluoride laser for CE analysis of a refined Southwest Asian heroin hydrochlor- ide exhibit [81].


antigen

-~ I C. CZE with LIF detection

I Minutes

Figure 9. Competitive immunoassay procedure [85] .

the sample containing unlabeled antigen. The sample may contain either a known amount of antigen in the case of standard curve generation (calibration) or an unknown amount in the case of a sample. The amount of labeled antigen is determined by the desired dynamic range, which is typically sixfold to tenfold and centered around the labeled antigen concentration [86]. An internal standard is added to the sample. After the mixture is thoroughly mixed, a predetermined limited amount of specific antibody is added. In the ensuing reaction labeled and unlabeled antigens compete for the limited amount of antibody, with the important result that the amount of unbound labeled antigen increases as the concentration of unlabeled antigen in the sample increases. The free-solution interaction of antibody and antigen is usually quite rapid, and CE analysis can be performed in 1-10 min after the reaction is initiated. The purpose of the CE analysis is to separate bound from free labeled antigen so that the area of the free labeled antigen peak can be determined. In this way, the concentration of unlabeled antigen can be interpolated from the standard curve of concentration versus area. The standard curve generated by the assay exhibits a sigmoidal shape, typical of concentration response after a semi-log trans- formation. At high concentrations of antigen most of the labeled antigen is displaced from the antibody binding sites and the signal of unbound labeled-antigen is large, whereas the signal of the labeled complex is low. At low concentrations of antigen the situation is reversed, most of the labeled antigen is complexed to the antibody and thus

the labeled antigen signal is low whereas the labeled complex signal is large. Only the linear range between the two extremes is used for quantitation. Determination of the Concentration is performed by interpolation from the semi- log curve.

3.2.2 Noncompetitive assay

If antibody determination in a sample is needed, then a known amount of fluorescently labeled antigens can be added to the sample. The labeled antigen will form a complex specifically with the antibody. If there is an excess of fluorescently labeled antigen, then the formation of complex will be quantitative and directly dependent on the amount of antibody present in the mixture. The CE separation will reveal two zones by LIF detection, one corresponding to the complex and the other to free fluorescently labeled antigen. If the complex is stable on the time scale of separation, then it should be possible to quantify the amount of antibody in the mixture based on the amount of complex formed and/or the decrease in the amount of free fluorescently labeled antigen.

3.2.3 Applications

3.2.3.1 Insulin quantitation The first study of the CE-LIF assay system was reported by Schultz and Kennedy [87] for the analysis of insulin. They compared noncompetitive and competitive immunoassays using an FITC-labeled insulin and an He-Cd 442 nm excitation laser. Rapid separations were obtained. The relative standard deviation was 6.3% for the complex peaks and 2.9% for the free FITC-insulin peak. The detection limit for insulin by this competitive assay was about 3 n~ or 420 zmol injected. Figure 10 (A) presents the rapid separation of a mixture of FITC-insulin and anti-insulin Fab; (B), shows the calibration curve of competitive assay; and (C) is the calibration curve for a noncompetitive assay. Approxi- mately the same results were obtained with both techniques. Later these authors developed an assay using FITC- derivatized insulin as a labeled antigen and the Fab fragment of an anti-insulin monoclonal antibody as the immunoreagent. Reproducibility and reliability of the assay were improved when capillary zone electrophoresis (CZE) capillaries were coated with a neutral hydrophilic polymer. The assay had an average relative standard deviation of 3.4% and a detection limit of 3 nM or about 6 fg injected. Using this type of assay, the insulin content of single islets of Langerhans was determined to be 35 k 4 ng, which is in good agreement with values found in the literature. An electropherogram is presented in Fig. 11 [88]. Tao and Kennedy [89] developed an on-line competitive immunoassay for insulin to monitor insulin concentration in a flowing stream. This system was used for flow injection analysis to determine the content of single islets of Langerhans. It could also be used for sensor-like monitoring ~ 9 1 .

3.2.3.2 Human growth hormone

Proteins have been widely studied using immonoassay CE- W 1901 or CE-LIF [91, 921. Shimura and Karger [93]

2786 F. Couderc, E. Causse and C. Bayle

A

Electrophoresis 1998, 19, 2111-279O

' 1 l 2 5

k t

0 20 40 60 TIME (sec)

m

$ Y

a m 0.60 n. -u a N

m 0.30 E L

.- -

0 0

a a

0.00 - 0.00 100.00 200.00

C [insulin], (nM) 1 .oo m

2 Y GI al a u 0.50 a, N m .- - 5 0 z

0.00 0.00 100.00 200.00 300.00

[F(ab)l, (nM)

Figure 10. (A) Rapid separation of mixture containing 50 IIM of FITC- insulin and 25 mi of anti-insulin Fab. Peaks 2, 3 and 5 are HTC-insulin. Peaks 1 and 4 are due to the formation of a complex of Fab with FITC- insulin in peaks 2 and 5. (B) Calibration curve for determination of Fab by the competitive assay. (C) Calibration curve for determination of Fab by the noncompetitive assay [87].

presented a sensitive microscale analytical procedure called affinity probe capillary electrophoresis. One of the two pieces, which can form a biospecific complex is labeled with a fluorescent dye. The affinity probe is used to detect

0 0.4

I

:2 ,

0.8 1.2

Time (minl

Figure 11. Competitive assay performed on single islet extracts. The solid electropherogram comes from a sample containing 100 n~ FITC-insulin (peak 2), 100 n M Fab and 50 n~ internal standard (peak 1). The dashed electropherogram is from an extract of single islet [MI.

the analyte as a complex after separation of the excess free probe by capillary electrophoresis. As an example, a Fab fragment from a mouse monoclonal antibody of human growth hormone (HGH) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group (Fig. 12A, [93]). Samples were mixed with the purified labeled antibody fragment, and the associated complex was separated by capillary isoelectric focusing with LIF detection. Methionyl recombinant HGH was determined at the detection limit level of 5 X M. Mono- and dideamidated variants of methionyl recombinant HGH were detected simultaneously with the nondeaminated form of the antigen as separate peaks (Fig. 12B).

3.2.3.3 Morphine, phenyclidine

These two drugs were analyzed using a multianalyte drug assay (competitive assay) in urine using CE-LIF [94]. Morphine derivatized with Cys5 and Cys 5.5 (Biological Detection Systems, Pittsburg, PA, USA) was used to tag phenyclidine. The laser was an He-Ne 632.8 nm; a homemade LIF detector was used. Figure 13 [94] shows the electropherograms for different amounts of the two drugs. The relative electrophoretic mobilities of Cys 5-morphine and Cys 5.5-phenyclidine are shown in Fig. 13 [94]; Cys 5 in the mixture acts as a reference peak [95].

3.2.3.4 Digoxin

Digoxin was analyzed using CZE. The tracer was labeled with B-phycoerythrin [96, 971. The electropherograms of different concentrations of this drug in human serum are presented in Fig. 14 [96]. A four-point calibration curve obtained by the authors is also reprinted.

Electrophoresis 1998, 19, 2111-2790

0

Drug analysis by CE-LIF 2787

I 2D C

A A

A

Mcruptocthylamioc

CootroUcd ar idat ion

Tctramcthylrhodaminc iodoacctamidc

Pepsin

___,

gab'

Mooothiol Fnb'

IR-Fab'

Figure 12. (A) Preparation of labeled antibody fragment. (B) Simultaneous detection of met-rbGH variants. An artificial mixture of met-rhGH, the monodeamidated variant (N149D) and the dideamidated variant (N149D, N152D), each at a concentration of 10 ng/mL, was subjected to affinity probe capillary electrophoresis [93].

3.2.3.5 Theophylline, ethosuximide, paracetamol,

For drugs whose concentrations are too low for detection or that coelute with endogeneous compounds (such as proteins) coming from a biological sample, it is necessary before sample injection to add a sample preparation step which includes drug extraction or removal of proteins by ultrafiltration or precipitation. Steinmann and Thormann [95] recently developed a competitive immunoassay for simultaneous quantitation of theophylline, ethosuximide, paracetamol, salicylate, and quinine using fluorescent

salicylate, and quinine

2 ai 1 IC C

A - A

Figure 13. Electrophergrams demonstrating titration of labeled morphine and phencyclidine (PCP) with antisera. (A) Peak 1, Cy5.5-F'CP, 2, CyS- morphine; R, CyS diacid used as internal standard at 10, 10,2 and 2 nmoV L, respectively. (B) Mixture in A plus antiserum to morphine. Peak C, CyS- morphme-antibody complex. (C) Mixture in A plus antiserum to PCP. (D) Mixture in A plus both antisera [94].

tracers, MEKC, and LIF detection. The originality of their study was the use of commercial tracers from fluorescence polarization immunoassay kits from Abbott (Abbott Labo- ratories, Abbott Park, IL, USA). Figure 15 [95] presents the simultaneous determination of paracetamol and salicylate. Tracer and antibody reagents for paracetamol and salicylate were mixed with serum prior to analysis. The electropherogram presented in panel A depicts the data issued from a mixture containing the zero level calibrators of both reagent kits. The free paracetamol traced (Tp 8.3 min), the paracetamol antibody-tracer complex (Cp 10 min), and the salicylate antibody-tracer complex (Cs 10.5 min) were monitored as sharp peaks whereas the free salicylate tracer appeared as a broad double peak after 10.73 min. Application of the calibrator instead of a blank (B-D) will further reveal the independent performance of the two immunoassay s .

Beside immunoassay affinity, affinity capillary electrophoresis (ACE) was used to quantitate sulfonamide drugs in body fluids. Tim et al. [98] proposed a direct method to determine picomolar tight binding sulfonamide drug concentrations in human urine and plasma. Fluorescein-labeled human carbonic anhydrase I1 was used as a fluorescent reagent to detect such drugs. The assay was performed by adding a known amount of labeled human carbonic anhydrase 11 to a sample containing an unknown amount of target drug. The amount of drug bound to human carbonic anhydrase I1 is determined by CE-LIF using a charged competitive ligand that strongly shifts the migra- tion of human carbonic anhydrase I1 unless it is blocked by the sulfonamide drug. The dorzolamide detection limit was 16.5 PM in aqueous solutions and 62.5 PM in biological fluids.

3.2.3.6 Vitamin A Ma et al. [99] performed a fast microassay of serum vitamin A. An He-Cd laser set at 325 nm was used for excitation,

2788 F. Couderc, E. CaussB and C. Bayle

D-bphyco L Ab comp1.x

A

B

C

D

I 0 I 2 3 4 5 6 1 9

lime in m ~ n m ~ i

B PO , I

5 0 2 4 6 8 10 12

Dig& mncntration. ng/d

Figure 14. (A) Electropherogram of CE-LIF of digoxin assay mixture with serum calibrators. Peak 1, antigen-antibody complex of the Fab and digoxin-B phycoerythrin (D-BP); 2, D-PB; 3, unknown fluorescent substance derived from serum. (A) 0.42 nglmL digoxin; (B) 2.72 nglrnL digoxin; (C) 5.21 ng/mL digoxin; (D) 10.42 ng/mL digoxin. (B) Calibration curve for digoxin assay by CE-LIF [97].

and the fluorescence of the vitamin A-retinol binding protein complex was monitored. About 8 nL of serum sample were injected for direct analysis without any sample preparation. Subfemtomole levels of vitamin A in human blood could easily be detected. A virtual relationship was obtained for the retinol concentration for 28 serum samples, 19 dried venous blood samples, and nine capillary dried blood samples collected on filter paper. The absolute detection limit for retinol was below 3 p g L [loo].

4 Conclusion

In this review the different studies involving the use of CE- LIF for drug analysis were examined. Even though a large

-0.1 - Electrophoresis 1998, 19, 2171-2190

.:,F:: 4 3 Kt 5 0.4

CP CP

-0.1 7.5 9.0 10.5 12.0 7.5 9.0 10.5 12.0

Time lrninl Time [mini

Figure 15. MEKC of salicyclate and paracetamol. Electropherograms obtained with a mixture containing solution S (antibody preparation) and solution T (fluorescein labeled drug) and serum calibrator. (A) Zero calibrator of both drugs; (B) 100pglmL salicylate and 20 pg/mL paracetarnol; (C) zero calibrator of salicylate and 20 pg/mL paracetamol, and (D) 100 pg/mL salicylate and zero calibrator of paracetamol. Ts, free salicylate tracer; Tp, free paracetamol tracer; Cs, salicylate antibody-tracer complex; Cp, paracetamol antibody-tracer complex [95].

chemical functions exists, very few studies about drugs have been performed with the derivatization of the molecule. Only immunoassays and native fluorescence were used. A promising approach using laser-induced resonance energy transfer to achieve high sensitivity detection for drugs has been proposed recently [loll. An important aspect of this kind of detection is that the analytes do not need to be fluorescent or made fluorescent. Ideally, the acceptor molecule should be soluble in an aqueous buffer, have minimal absorbance at the maximum absorption of the analyte, show fluorescence only in the presence of the analyte molecule, and be capable of accepting energy with high efficiency from a wide range of substances. Terbium and Europium are the ideal elements for fitting the criteria of an ideal acceptor. The authors studied aspirin and its metabolite monitoring Tb3+ luminescence at 547 nm. They increased the sensitivity of conventional detection by 1000, even if the LOD remains quite high, within the M range. The increased popularity of capillary electrophoresis and of the different LIF detectors commercially available, with more and more laser wavelengths to be used, will enhance the increased development of drug assays with CE- LIF in the future.

Received May 18, 1998

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Drug analysis by capillary electrophoresis and laser-induced fluorescence

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