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.