Application of multisegment injection on quantification of creatinine and standard addition analysis of urinary 5‐hydroxyindoleacetic acid simultaneously with creatinine normalization

Abstract

Application of multisegment injection on quantification of creatinine and standard addition analysis of urinary 5-hydroxyindoleacetic acid simultaneously with creatinine normalizationIn this paper, the development of a simple dilute-and-shoot method for quantifying urinary creatinine by CE-ESI-MS was described. The creatinine analysis time was about 7 min/sample by conventional single injection (SI) method and can be significantly reduced to less than 2 min/sample with multi-segment injection (MSI). In addition, the standard addition analysis of 5-hydroxyindole-3-acetic acid (5-HIAA) and creatinine normalization was performed within one run by the MSI technique, and the total analysis time was 14-min faster compared to the SI method for analyzing the same set of samples. The uses of isotopic and non-isotopic internal standards (ISs) were compared. Creatinine-(methyl-13 C) and 5-hydroxyindole-4,6,7-D 3 -3-acetic-D 2 acid (5-HIAA-D 5 ) used as isotopic ISs can provide both accurate and precise results. In contrast, 1,5,5-trimethylhydantoin (1,5,5-TH) used as the non-isotopic IS for creatinine may cause a bias of over 13% in SI method and even worse when the MSI technique was used. Another compound, 2-methyl-3-indoleacetic acid (2-MIAA), was determined not suitable for MSI analysis of 5-HIAA due to endogenous interferences despite its acceptable performance in conventional methods of analysis.Abbreviations: 1,5,5-TH, 1,5,5-trimethylhydantoin; 2-MIAA, 2methyl-3-indoleacetic acid; 5-HIAA, 5-hydroxyindole-3-acetic acid; IS, internal standard; MRM, multiple reaction monitoring; MSI, multi-segment injection; RPA, relative peak area; SI, single injection variations due to water intake or diet composition. This gives rise to the difficulty of interpreting urinary metabolite concentrations and necessitating urine volume calibration [2,3].The conventional urine normalization approaches include flow rate correction, measurement of urine osmolality or specific gravity, and the commonly used creatinine correction [2,3]. The daily renal excretion of creatinine via glomerular filtration remains at a relatively constant rate under normal conditions [2-4], and the urinary creatinine level has been found relatively stable when the urine secretion rates are above 1 mL/min [3,5]. Despite the potential influence of age, gender, diet intake, and other factors, creatinine normalization is still widely applied and generally accepted in metabolomic studies for correcting solute concentrations of urine samples [1][2][3]. Furthermore, urinary creatinine levels are also measured for the assessment of renal function in Color online: See the article online to view Figs. 1-4 in color.

Capillary Electrophoresis: A Three-Year Literature Review

“…The merits of using organic solvents as modifiers/solvents have been demonstrated in a number of publications. The wide selection of organic solvents, with their distinct physicochemical properties, broadens the scope of potential applications of CE techniques. − …”

High‐throughput capillary electrophoresis analysis of biopharmaceuticals utilizing sequential injections

“…Further, in the present study, due to the voltage-assisted separation between injections and short injection lengths, band broadening was minimized. This is an advantage over the more commonly used multisegment method in which long BGE spacers are hydrodynamically injected [23]. Such long injection pressures often lead to parabolic flow profile and peak broadening.…”

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…Besides manipulating the EOF [402], the (relative) low-throughput associated with individual runs can be addressed by injecting multiple segments of sample between BGE zones, allowing the components to migrate into those spacers [403]. This approach has been used not only to decrease the analysis time of creatinine (by approximately 70%) [404,405] but also to inject multiple serum filtrate samples for metabolomic studies, including those of women in the last trimester of their pregnancy [406]. Other applications of this approach have been also recently described [407][408][409][410], noting that current analytical constraints of the approach include poor concentration sensitivity, potential loss in isobar/isomer resolution, sample carryover, its restriction to ionic metabolites [403], as well as the need to perform extensive method optimization to improve the compatibility with NACE protocols [411].…”