Applicability of NACE

In CE the separation of solutes is due to differences in the charge over size ratios and thus very similar substances may be difficult to separate in aqueous CE unless special mechanisms like micellar elec-trokinetic chromatography (MEKC) are involved. Of course this involves addition of one or more surfactants.

Figure 3 Electropherograms of cis-trans- and diastereo-isomers. (A) Separation of cis- and trans-flupenthixol decanoate using 50 mmol L_1 ammonium acetate and 1 mol L_1 acetic acid in methanol + acetonitrile (1:1, v/v), above: i-flupenthixol decanoate with 0.5% trans-isomer added; below: trans-flupenthixol decanoate. Conditions: 64 cm (55.5 cm to the detector) x 50 |im i.d. capillary, injection for 3 s at 5 kPa (50 mbar), 25°C, 30 kV (9 |jA) and UV detection at 230 nm. Test solution: 5.0 mg mL_1 of the sample in methanol + acetonitrile (1 : 1 v/v). Peak identity: 1, cis-flupenthixol decanoate; 2, trans-flupenthixol decanoate; U, unknown. (B) Separation of dipeptides (diastereomers); (C) separation of quinine and quinidine (diastereomers). Conditions as in (A) with a detection wavelength of 214 nm. Adapted with permission from Hansen SH, Bjcrnsdottir I and Tj0rnelund J (1997) Separation of cationic cis-trans (Z-E) isomers and diastereomers using nonaqueous capillary electrophoresis. Journalof ChromatographyA 792: 49-55.

Figure 3 Electropherograms of cis-trans- and diastereo-isomers. (A) Separation of cis- and trans-flupenthixol decanoate using 50 mmol L_1 ammonium acetate and 1 mol L_1 acetic acid in methanol + acetonitrile (1:1, v/v), above: i-flupenthixol decanoate with 0.5% trans-isomer added; below: trans-flupenthixol decanoate. Conditions: 64 cm (55.5 cm to the detector) x 50 |im i.d. capillary, injection for 3 s at 5 kPa (50 mbar), 25°C, 30 kV (9 |jA) and UV detection at 230 nm. Test solution: 5.0 mg mL_1 of the sample in methanol + acetonitrile (1 : 1 v/v). Peak identity: 1, cis-flupenthixol decanoate; 2, trans-flupenthixol decanoate; U, unknown. (B) Separation of dipeptides (diastereomers); (C) separation of quinine and quinidine (diastereomers). Conditions as in (A) with a detection wavelength of 214 nm. Adapted with permission from Hansen SH, Bjcrnsdottir I and Tj0rnelund J (1997) Separation of cationic cis-trans (Z-E) isomers and diastereomers using nonaqueous capillary electrophoresis. Journalof ChromatographyA 792: 49-55.

Table 3 Applications of NACE in analysis of food, pharmaceuticals and biological fluids

Solvents

Electrolytes

Analytes

Applications within food NMF-dioxane (1 : 1 v/v)

Propylene carbonate

Applications within pharmaceuticals 10-100% MeOH

Mixture of MeOH and H2O MeOH

MeOH and mixture of MeOH and MeCN

MeOH, MeCN, mixture of MeOH and MeCN, formamide, NMF, DMF, DMA, DMSO

NMF, formamide and mixtures of both NMF

MeOH

MeCN

Formamide

40 mmol L 1 Tris, 2.5 mmol L 1 anthraquinone-2-carboxylic acid

500 mmol L_1 magnesium acetate tetrahydrate

Tetraalkylammonium ions, long chain trimethylammonium ions 20 mmol L~1 tetradecylammonium bromide (vitamin K1 and preservatives)

Ammonium acetate, acetic acid

Ammonium acetate, acetic acid 5 mmol L~1 ammonium acetate, 100 mmol L~1 acetic acid

Ammonium acetate, tetrabutylammonium bromide, tetrabutylammonium hydrogen sulfate and tetrapentylammonium bromide 25 mmol L~1 ammonium acetate, 0-1 mol L~1 acetic acid or 100 mmol L~ sodium acetate Application: 25 mmol L~1 ammonium acetate, 1 mol L~1 acetic acid in MeCN

Free saturated long chain fatty acids (n-C14-n-C26). Separation of dimeric and trimeric acids and hydrogenated fish oil Tetracycline (TC), oxytetracycline (OTC), chlorotetracycline (CTC), demeclocycline, 4-epitetracycline, anhydrotetracycline, 4-epianhydrotetracycline, and desmethyltetracycline TC, OTC and CTC in milk and plasma Phenanthrene, ß-naphthol, preservatives: methylparaben, ethylparaben and propylparaben, thiourea (EOF marker) and vitamin K1

Haloperidol and synthetic putative metabolites, pyrazoloacridine and mifentidine Haloperidol, cimetropium and mifentidine Haloperidol and its synthetic putative metabolites, pyrazoloacridine and its synthetic putative metabolites, mifentidine and its synthetic putative metabolites Tamoxifen and four phase I metabolites

25 mmol L~1 ammonium acetate, 10 mmol L~1 citric acid and 118 mmol L~1 methanesulfonic acid

20 mmol L~1 ammonium acetate,

1 mol L~1 acetic acid Citric acid or acetic acid mixed with Tris. Chiral selectors: ß-CD, y-CD and derivatized ß-CD. Addition of long chain alkyl ammonium salts investigated

Ammonium acetate, acetic acid, quinine

Imipramine, di-desmethylimipramine, desmethylimipramine, methylimipramine and imipramine-N-oxide. Maprotiline, amitriptyline, litracene, protriptyline and nortriptyline. Application: imipramine N-oxide and impurities Tetracycline and three degradation products. Tetracycline, oxytetracycline, doxycycline, desmethyltetracycline and chlortetracycline Morphine analogues, antihistamines, antipsychotics and stimulants Racemic mixtures of chlorphedianol, chlorcyclizine, ethopropazine, mianserin, nefopam, primaquine, propiomezine, trihexyphenidyl, trimeprazine, trimipramine and thioridazine NaCl Dansylated amino acids 1 Dansylated amino acids

A/-3,5-dinitrobenzylated amino acids, ( ± )-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate and

A/-[1-(1-naphthyl)ethyl]phthalomic acid Atenolol, bisoprolol, bunitrolol, metroprolol, pindolol, propranolol, salbutamol, ephedrine, epinephrine, cisapride and synthetic impurities

Tetra-n-butylammonium perchlorate. Chiral 1-Naphthylethylamine, 1-phenylethylamine, selector: ( # )-18-crown-6-tetracarboxylic phenylalanine, DOPA, tryptophan, acid norephedrine, noradrenaline and

2-amino-1,2-diphenylethanol

( $ )-Camphorsulfonic acid potassium or sodium salt, 1 mol L~1 acetic acid 0.2 mol L_1 Tween 20

Table 3 Continued

Solvents

Electrolytes

Analytes

Formamide, NMF, DMF, DMA, DMSO, MeOH, MeCN and mixtures of MeOH and MeCN MeOH : MeCN (75 : 25)

Mixtures of MeOH and MeCN

Mixtures of MeOH and MeCN MeOH

Applications within biological fluids 10-100% MeOH in H2O

MeOH

25 mmol L 1 ammonium acetate, 1 mol L_1 acetic acid

25 mmol L_1 ammonium acetate,

1 mol L_1 acetic acid

500 mmol L_1 magnesium acetate tetrahydrate Ammonium acetate, ammonium chloride, acetic acid, trifluoroacetic acid, formic acid, methane sulfonic acid

Sodium acetate

20 mmol L~1 CAPS and 0-40 mmol L~1 Brij 35

20 mmol L~1 ammonium acetate,

1 % acetic acid 500 mmol L~1 magnesium acetate tetrahydrate

5 mmol L~1 ammonium acetate,

100 mmol L~1 acetic acid 50 mmol L~1 ammonium acetate, 159 mmol L~1 sodium acetate and 0.002% (w/v) hexadimethrine bromide

Morphine, codeine, normorphine, thebaine, noscapine and papaverine. Application: morphine in opium tincture Morphine

Oxytetracycline in an ointment

Cis-trans (Z-E) isomers of chlorprothixene, thiothixene, clopenthixol, flupenthixol, flupenthixol decanoate, clomiphene and diastereomers: L-Ala-L-Phe, L-Ala-D-Phe; quinine, quinidine, cinchonine and cinchonidine A range of penicillins, cephalosporins and nonsteroidal anti-inflammatory drugs Mesoporphyrin, coporphyrin, pentaporphyrin, hexacarboxylporphyrin, heptacarboxylporphyrin and uroporphyrin

Pyrazoloacridine, two metabolites and a synthetic degradation product in urine Tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), demeclocycline, 4-epitetracycline, anhydrotetracycline, 4-epianhydrotetracycline and desmethyltetracycline. TC, OTC and CTC in cow milk and human plasma Mifentidine and three metabolites in rat liver homogenate Acetylsalicylic acid and three metabolites: salicylic acid, salicyluric acid and gentisic acid in plasma and urine

Reproduced with permission from Bjornsdottir etal. (1998) Electrophoresis 19: 2179.

NACE provides high separation power of very similar substances without using additives like surfactants or cyclodextrines. In Figures 1 and 2 the separation of very similar substances using NACE are compared to separation in an aqueous CE and a MEKC system. As seen in Figure 2, even substances expected to have identical mass over charge may be separated in a short time compared to the aqueous systems. Figure 3 shows the separation of cis-trans isomers and diastereoisomers. These isomers are also expected to have the same mass over charge ratio. The use of NACE in the analysis of food, pharmaceuticals and biological fluids has been reviewed by Bj+rnsdottir and co-workers and in Table 3 an overview of applications is given. An important practical consequence of using a NACE separation medium is that the organic phases resulting from either simple extractions or from eluents from solid-phase extractions can be injected directly into the system, thereby saving time.

Furthermore, some NA solvents seem promising for CE-MS experiments due to the volatility of the solvents and the relatively low current generated in the organic solvents. The low current is comparable to the current generated in electrospray MS interfaces and therefore the stability of online CE-MS is optimized.

Two questions are often raised in connection with practical work with NACE. How important is it that the electrophoresis medium is really nonaqueous? This is not crucial. A content of water up to 1% will not influence the separation efficiency and selectivity significantly. Is it possible to perform quantitative analysis using NACE? Yes, if steps against evaporation are taken when volatile solvent are used, the reliability of the methods is comparable to that of aqueous systems (Table 4). A number of applications including validated quantitative methods are given in Table 4.

Table 4 Validation data from quantitative NACE analysis of food, pharmaceuticals and biological fluids

Analytes

Linearity (r2)

Repeatability of inj. ( % RSD) LOD

Accuracy

Free saturated long chain 0.994 (n16-n20)

Tetracyclines in plasma and 0.999 milk

Vitamin K1, propylparaben 0.993 and methylparaben

Tetracycline and three 0.993-0.998 degradation products

Oxytetracycline in an > 0.999 ointment

Morphine in pharmaceutical > 0.999 products

Acetylsalicylic acid and three Good in the range: metabolites: salicylic acid, 5-500 ^g mL~1 salicyluric acid and gentisic acid in plasma and urine

Inter-day: 2.1-39% (n = 6) at three concentration levels

Inter-day repeatability of the 25 ng mL 1 of TC, method: 3.6-10.2% (n = 6) OTC or CTC at three concentration levels

- 3% (n = 6) at three nd concentration levels for all three analytes

Inter-day: 3.4-13% (five concentration levels)

Inter-day: 2.8-4.4% for peak nd area (n = 6) at three conc. levels. For migration time: > 0.8% within day (n = 8) and < 3.3% in-between days (n = 6)

Inter-day: plasma: 0.8-5.0% LOQ : 5 ^g mL~1 (n = 6), urine: 1.0-5.4% in plasma and (n = 6) at three conc. levels 25 ^g mL~1 in urine nd

97.2% Oxytetracycline (n = 6, %RSD = 4.2%) and 63.3% (n = 6, %RSD = 3.6%) at two conc. in milk

98% Vitamin K1 (n = 6, %RSD = 4.95%) 96% Propylparaben (n = 6, %RSD = 4.45%) 82% Methylparaben (n = 6, %RSD =2.26%)

96.1-97.3% Oxytetracycline at three concentration levels (n = 6)

100.7-101.2% (three concentrations)

nd, not determined; LOQ, limit of quantitation. Reproduced with permission from Bj+rnsdottir et al. (1998) Electrophoresis 19: 2179.

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