Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and ß-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and ß-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the ß-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

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.

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-ß-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.

Hemolysis interference in 10 coagulation assays on an instrument with viscosity-based, chromogenic, and turbidimetric clot detection.

INTRODUCTION: Hemolysate in plasma samples from patients may cause misleading results in coagulation assays. Even though modern coagulation instruments often are equipped with modules that can detect hemolysis, icterus, and lipemia (HIL), studies that report the influence of these interferences are still limited. The present paper focuses on the influence of hemolysis on 10 coagulation assays. METHODS: Artificial hemolysis was created by freezing/thawing, and the hemolysates generated were added to pools of patient plasma. Pathological and normal levels were pooled separately. These spiked samples were analyzed on a STA R Max 2 instrument. The coagulation assays evaluated utilize clot, chromogenic, or immunoturbidimetric detection. RESULTS: Four of the evaluated assays were not influenced by hemolysis: fibrinogen, von Willebrand factor antigen, activated partial thromboplastin time, and factor VIII. Interestingly, normal and slightly elevated prothrombin time (INR < 2.0) was insensitive to hemolysis, whereas samples with a high INR (≥2.0) exhibited falsely high readings. The assays for antithrombin and fibrin D-dimer displayed an intermediate sensitivity to hemolysis. The most sensitive assay turned out to be anti-Xa, followed by protein C and protein S. For the anti-Xa assay, the results are decreased by 10% already at 0.5 g/L hemoglobin. CONCLUSION: The present study shows that hemolysis affects several of commonly used coagulation assays. Since the sensitivity for hemolysis is dependent on the brand of the assay as well as the instrument and principle of measurement, it is necessary to evaluate the influence of each specific combination.

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

1S,4R-(+)-ketopinic acid [(+)-KPA] has been introduced as a chiral selector for the separation of pharmacologically active amines by non-aqueous capillary electrophoresis (NACE). (+)-KPA gave enantioresolution for most of the compounds previously separated by 2R,3S,4R,5S-(-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid [(-)-DIKGA], but with a reversed migration order. A complete enantioresolution (Rs=4.2) was obtained for timolol, a compound that could not be resolved using (-)-DIKGA as the selector. Thus, (+)-KPA was evaluated for the enantiomeric purity determination of S-timolol. A method based on pre-concentration by transient isotachophoresis (tITP) provided a limit of detection (LOD) of 0.2% R-timolol in S-timolol samples. Because of the lack of enantioresolution of ephedrine when (+)-KPA was used as the selector, a method with (-)-DIKGA has been developed and validated for determination of the enantiomeric purity of the 1R,2S enantiomer. The method gave good precision and accuracy with an LOD (S/N=3) of 0.033% for the enantiomeric impurity 1S,2R-ephedrine.

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.

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.

Effect of alkali metal hydroxides on the enantioseparation of amines using di-O-isopropylidene-keto-L-gulonic acid as the selector in NACE.

The present work demonstrates the importance of the ionic composition in the BGE for enantioseparation. (-)-2,3:4,6-di-O-Isopropylidene-2-keto-L-gulonic acid ((-)-DIKGA) has been used as the chiral selector in methanolic and ethanolic BGEs. The influence of added alkali metal hydroxides on the EOF and the chiral separation of amines (atenolol, isoprenaline, pindolol and propranolol) have been studied. The ion-pair formation constants in ethanol were determined by precision conductometry for the enantiomers of pindolol with (-)-DIKGA, for Li(+), Na(+) and Cs(+) with (-)-DIKGA, and also for the corresponding alkali metal hydroxides. The effective mobilities and the enantiomeric mobility differences were affected by the type of alkali metal hydroxide (LiOH, NaOH, KOH, RbOH or CsOH) added to the BGE. The effective mobility and mobility difference were increased with decrease in solvated radius of the alkali metal cation. These differences could partly be correlated to the ion-pair formation constants of the alkali metal cations with the chiral selector, affecting the equilibrium concentration of the free selector. The electroosmosis was also affected by the alkali metal hydroxide added to the BGE. The cathodic electroosmosis decreased with decreasing solvated radius of the alkali metal cation added to the BGE. Interestingly, the cathodic EOF was even reversed, i.e. became anodic in the ethanolic BGEs containing KOH, RbOH or CsOH and the methanolic ones with RbOH and CsOH.