Page 39 - Clinical-Medical Solution for Clinical Chemistry

Determination of Unbound Urinary Amino Acids Incorporated
with Creatinine Normalization by LC-MS/MS Method with
CLAM-2000 Online Sample Pre-treatment

Table 1: Analytical conditions of twenty two amino acids without
derivatization on LCMS-8040 and CLAM-2000

Column : Intrada Amino Acid (100 mmL x 3 mmID, 3µm)
Flow rate : 0.6 mL/min
Mobile phase : A: ACN / THF / 25mM ammonium formate / FA = 9 / 75 /16 / 0.3 (v)
B: ACN / 100mM ammonium formate = 20 / 80
Elution mode : Gradient elution, 0-3min (0% B) → 9min (17% B) →
16-18.5min (100% B) → 19min (0% B)
Oven temp. : 35°C
Injection vol. : 5.0 µL
Interface : ESI
MS mode : Posi, MRM
Block temp. : 400°C
DL temp. : 250°C
CID gas : Ar (230kPa)
Nebulizing gas ow : N 2, 2 L/min
Drying gas ow : N 2 , 15 L/min

Results and Discussion

Quantitative method for 22 AA and CRE on CLAM-LC-MS/MS platform

The 20 proteinogenic amino acids (AA), citrulline (Cit) and MRM chromatograms of the 22 AA and CRE mixed
ornithine (Orn) as well as creatinine (CRE) are the standards obtained on CLAM-LC-MS/MS platform. For
targeted analytes in urine in this study. This is because calibration curve construction, a calibrant series of eight
citrulline and ornithine are the dietary amino acids in the levels (0.1, 0.5, 1, 2, 5, 10, 50 and 100 µM) were
urea cycle along with arginine. A MRM method for prepared and analysed. A few selected calibration curves
quantitative analysis of the 22 AA and CRE (creatinine) by IS method are shown in Figure 3. The accuracy and
was established with IS (internal standard) method as repeatability (based on area) of the method with 10 uM
summarized in Table 2. In a previous study [5], it was mixed standards are shown in Table 2 and the results
observed that glycine exhibited low peak intensity and obtained with urine samples (not shown in the table) are
sensitivity in MRM mode (76.1>30.1). Thus, SIM mode also satis ed.
(m/z 76.1) was selected for glycine. Figure 2 shows the

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