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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 β-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 β-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 β-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.

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The aim of this study was to evaluate and compare some compounds as EOF markers in aqueous and non-aqueous background electrolytes AIMThe aim of this study was to evaluate and compare some compounds as EOF markers in aqueous and non-aqueous background electrolytes (BGEs). Different electrolyte system as well as BGEs with chiral selectors and micellar additives has been investigated. Short Code 316301It is always necessary to know the electro-osmotic flow (EOF) and the reproducibility of the EOF during method Influence of pH and buffer type Sodium borate buffer Ammonium acetate buffer IntroductionShort Code 316301 (EOF) and the reproducibility of the EOF during method development or troubleshooting in CE and sometimes it can be important to more accurately determine the EOF. Sodium borate bufferAll markers seem to be useful in borate buffer except:All analytes seem to be useful in acetate buffer except:• MBD at pH 8.0 and 9.0 (lower µ obs than the other) • Diphenyloxazole at pH 8.0 and 9.0 The most common was to measure the EOF is through the injection of a neutral marker. x• Iohexol who is known to complexate with borate, and was used as a marker for complexation.• Anthracene that differed in borate buffer at pH 8, 9 and 10. obs • Diphenyloxazole at pH 8.0 and 9.0 As expected, the RSD(%) for µ obs for all markers within one pH is higher at pH 4-6 (1-4%) than for pH 8-9 (0.5-0.9 %) There is a lack of a more general comparative studies of EOF markers in the literature. The previously published comparative studies only include one aqueous system at a specific pH [1] and micellar systems [2].• Anthracene that differed in borate buffer at pH 8, 9 and 10.• MBD had a lower observed mobility (µ obs ) in borate buffer. Interestingly, the decrease is more pronounced at pH 10.pH is higher at pH 4-6 (1-4%) than for pH 8-9 (0.5-0.9 %)