Ylva Hedeland

Development of a chiral non-aqueous capillary electrophoretic system using the partial filling technique with UV and mass spectrometric detection.

A chiral non-aqueous CE system with UV and mass spectrometric detection has been developed. The enantioseparation was promoted by diastereomeric complex (ion-pair) formation between the amines (e.g. salbutamol, atenolol) and the chiral selector, (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid [(-)-DIKGA]. Different solvent mixtures were studied, as well as different concentrations of (-)-DIKGA and ammonium acetate in the background electrolyte. A partial filling technique was developed with a selector plug composed of (-)-DIKGA and ammonium acetate in a solvent mixture of methanol and 2-propanol. The separated enantiomers of pronethalol were detected by a Q-TOF MS system equipped with a sheath-flow electrospray ionization interface.

Ketopinic acid and diisoproylideneketogulonic acid as chiral ion-pair selectors in capillary electrophoresis. Enantiomeric impurity analysis of S-timolol and 1R,2S-ephedrine.

Three chiral selectors (diketogulonic acid, benzoxycarbonylglycylproline and ketopinic acid) have been introduced for enantioseparation of pharmacologically active amines in non-aqueous capillary electrophoresis. The use of organic solvents, instead of aqueous buffers in the background electrolyte facilitated ion-pair formation between the analytes and the chiral selectors. The enantioresolution was strongly affected by the choice of selector and organic solvent but also depended on the other electrolytes. The most important parameter for the enantioresolution, apart from the choice of chiral selector, was the direction and magnitude of the electro-osmosis. Thus, covalently coated capillaries were used to suppress and to reverse this flow. Furthermore, the alkali metal hydroxide added to the background electrolyte had a great influence on the electro-osmosis. Exchanging LiOH for NaOH, was found to decrease the electro-osmotic flow. Interestingly, the flow was altered from cathodic to anodic, with KOH, RbOH or CsOH added to the ethanolic BGE. The occurrence of a reversed electro-osmosis had a great positive effect on the enantioresolution. An appropriate choice of solvent and electrolytes promoted also fast chiral separations, e.g., the enantiomers of isoprenaline were resolved within one minute. The capillary electrophoresis systems developed within this work were applied for enantiomeric purity determinations of different pharmaceutical forms of drug products. A detection limit of 0.033 % was achieved for 1S,2R-ephedrine, the enantiomeric impurity in Efedrin®, when diketogulonic acid was used as the selector. By using the pre-concentration technique, transient isotachophoresis, the peak efficiency was enhanced for the enantiomers of timolol. This facilitated the introduction of a higher concentration of the sample into the capillary electrophoretic system containing ketopinic acid as the selector, and lowered the detection limit from 2.5 % to 0.2 % for the enantiomeric impurity R-timolol compared with injection without transient isotachophoresis. The volatility of the non-aqueous media in capillary electrophoresis facilitated the hyphenation to mass spectrometry. The partial filling technique ensured that the selector did not contaminate the mass spectrometer, and the separated enantiomers of e.g., pronethalol were detected in the selector-free zone.

Chiral separation of amines with N-benzoxycarbonylglycyl-L-proline as selector in non-aqueous capillary electrophoresis using methanol and 1,2-dichloroethane in the background electrolyte.

N-Benzoxycarbonylglycyl-L-proline (L-ZGP) has been introduced as a chiral selector for enantioseparation of amines in non-aqueous capillary electrophoresis. Methanol mixed with different proportions of dichloromethane, 1,2-dichloroethane or 2-propanol containing L-ZGP and ammonium acetate was used as the background electrolyte. Enantioseparation of different types of pharmacologically active amines was performed, e.g. the local anaesthetic bupivacaine and the beta-adrenoceptor blocking agent pindolol. Addition of the solvents (dichloromethane, 1,2-dichloroethane or 2-propanol) gave an improved chiral separation partly due to a distinct decrease in the electroosmotic flow. The use of 1,2-dichloroethane in the background electrolyte gave higher precision in migration time (RSD 2.2%) compared to the systems containing dichloromethane. An enantiomeric separation of mepivacaine was performed within 72 s by use of short-end injection with an effective capillary length of 8.5 cm.

Evaluation of electroosmotic markers in aqueous and nonaqueous capillary electrophoresis

The most common method to determine the EOF in CE is to measure the migration time for a neutral marker. In this study, 12 compounds (three novel and some previously used) were investigated as EOF markers in aqueous and nonaqueous BGEs. In the aqueous buffer systems (ammonium acetate, sodium phosphate, and sodium borate) the evaluation included a wide pH range (2–12). Two BGEs contained chiral selectors (sulphated‐β‐CD, (−)‐diketogulonic acid) and one that contained a micellar agent (SDS) were included in the study. The majority of the evaluated compounds were found to migrate with the EOF in the water‐based BGEs and are thus useful as EOF markers. However, in the SDS‐based BGE only four of the compounds (acetone, acrylamide, DMSO, and ethanol) were found to be applicable. In the nonaqueous BGEs 11 markers (acetone, acetophenone, acrylamide, anthracene, benzene, 4‐(4‐methoxybenzylamino)‐7‐nitro‐2,1,3‐benzoxadiazole, benzyl alcohol, 2,5‐diphenyloxazole, ethanol, flavone, and mesityl oxide) seemed to be functional as EOF markers. Even though several of the evaluated compounds can be used as EOF markers in the investigated BGEs, the authors would recommend the use of acrylamide as a general marker for UV detection. Furthermore, the four fluorescent markers (of which three were novel) gave RSD values equal to the other markers and can be used for the determination of the EOF in CE or microchip CE with fluorescence detection.

Development of a capillary electrophoretic method for determination of plasma clearance of iohexol in dogs and cats

Renal function can be monitored by estimation of the glomerular filtration rate (GFR), for example, through measurement of the plasma clearance of a marker that is freely filtrated through the kidney without reabsorption. It has been proposed that iohexol is the most accurate marker for GFR determination in cats and dogs. However, there is a need for a validated capillary electrophoretic method that covers the concentration range for a full curve clearance estimate of iohexol. In the final method, the plasma samples were protein precipitated and the supernatant was analyzed in a background electrolyte containing borate buffer (0.06 m, pH 10.0). The method developed was proved to be linear (concentration range 18- 2900 mg/L) and had a good precision (e.g. 2.3-2.9% at 88 mg/L) and accuracy (e.g. 101-105% at 88 mg/L). Finally, the method was compared with a previously published and validated HPLC-UV method by parallel analysis of clinical plasma samples from dogs and cats administered Omnipaque®. This comparison showed excellent agreement between the two methods and no proportional or systematic error was observed. The proposed method is simple and has a low cost per sample, which makes it applicable for routine analysis.