LC-MS/MS Method for Serum Creatinine: Comparison with Enzymatic Method and Jaffe Method

<div><p>Accurate quantification of creatinine (Cre) is important to estimate glomerular filtration rate (GFR). Differences among various methods of Cre quantification were previously noted. This study aims to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for serum Cre and compare this method with clinical routine methods. LC-MS/MS analysis was performed on API 4000 triple quadrupole mass spectrometer coupled with an Agilent 1200 liquid chromatography system. After adding isotope-labeled Cre-d3 as internal standard, serum samples were prepared via a one-step protein precipitation with methanol. The LC-MS/MS method was compared with frequently used enzymatic method and Jaffe method. This developed method, with a total run time of 3 min, had a lower limit of quantification of 4.4 μmol/L, a total imprecision of 1.15%–3.84%, and an average bias of 1.06%. No significant matrix effect, carryover, and interference were observed for the LC-MS/MS method. The reference intervals of serum Cre measured by LC-MS/MS assay were 41–79 μmol/L for adult women, and 46–101 μmol/L for adult men. Using LC-MS/MS as a reference, the enzymatic method showed an average bias of -2.1% and the Jaffe method showed a substantial average bias of 11.7%. Compared with the LC-MS/MS method, significant negative bias was observed for the enzymatic and Jaffe methods in hemolytic and lipimic samples. We developed a simple, specific, and accurate LC-MS/MS method to analyze serum Cre. Discordance existed among different methods.</p></div

Method comparison of the serum Cre with (1) Deming regression and (2) Bland-Altman analysis of (A) LC-MS/MS versus enzymatic method, (B) LC-MS/MS versus Jaffe method, and (C) enzymatic method versus Jaffe method.

<p>Method comparison of the serum Cre with (1) Deming regression and (2) Bland-Altman analysis of (A) LC-MS/MS versus enzymatic method, (B) LC-MS/MS versus Jaffe method, and (C) enzymatic method versus Jaffe method.</p

Precision of serum Cre by LC-MS/MS analysis (low level was the pool of patient samples, middle level was 35.4 μmol/L Cre spiked in low level, and high level was 132.7 μmol/L Cre spiked in low level).

<p>Precision of serum Cre by LC-MS/MS analysis (low level was the pool of patient samples, middle level was 35.4 μmol/L Cre spiked in low level, and high level was 132.7 μmol/L Cre spiked in low level).</p

Stability of Cre under various conditions.

<p>Values are expressed as the bias from the initial concentration.</p

Chromatograms of (A) blank (20% methanol), (B) LLOQ (4.4 μmol/L, S/N = 921) and (C) a patient sample (43.9 μmol/L).

<p>Chromatograms of (A) blank (20% methanol), (B) LLOQ (4.4 μmol/L, S/N = 921) and (C) a patient sample (43.9 μmol/L).</p

Chromatograms of post-column infusion of Cre-d3 (132.7 μmol/L in 20% methanol) with a patient sample.

<p>Chromatograms of post-column infusion of Cre-d3 (132.7 μmol/L in 20% methanol) with a patient sample.</p

correlation analysis.

<p>The serum 25-(OH) D level correlates with the neutrophil apoptosis in the AECOPD patients(a). The serum 25-(OH) D level correlates with the phospho-p38 MAPK in the AECOPD patients(b). The correlation between the neutrophil apoptosis and phospho-p38 MAPK/p38 MAPK in AECOPD patients(c). The <i>r</i> and <i>p</i> values were assessed using Pearson's correlation analysis.</p

Baseline characteristics of the study sample, presented as the mean ± s.d. for continuous variable and as the percentage for categorical variables.

<p>^#smokers/ex-smokers/nonsmokers.</p><p>*FEV₁: Forced expiratory volume in 1 s.</p><p>†PaO₂: arterial oxygen tension.</p><p>^§LTDOT: Long-term domiciliary oxygen therapy.</p><p>^##Exacerbations requiring either hospitalization or treatment with oral antibiotics or oral steroids.</p><p>**Associations were tested with t-test and Chi-square.</p><p>Baseline characteristics of the study sample, presented as the mean ± s.d. for continuous variable and as the percentage for categorical variables.</p

1α,25VitD3 increased peripheral blood neutrophil apoptosis in the AECOPD patients.

<p>Neutrophils (1 ×10⁶) were pretreated with SB203580 (2.5 μM × 10^–5) for 0.5 h, followed by incubation with 1α,25VitD3 (6 ×10^–7 M) for 24 h. Neutrophil apoptosis was analyzed with flow cytometry (a). The right lower quadrant (RLQ) contains early apoptotic neutrophils with positive Annexin V-PE binding and negative 7-AAD binding (Annexin V-PE [z], 7-AAD [–]). The vital neutrophils in the lower left quadrant (LLQ) are Annexin V-PE (-),7-AAD (-). The upper right quadrant (RUQ) contains late apoptotic and necrotic neutrophils (Annexin V-PE [z], 7-AAD [z]). The amount of neutrophil apoptosis in the RUD and RLD quadrants are shown in (b), expressed as the % of the total cell count (10,000).The experiments shown are representative of at least 3 experiments. The data are presented as the means ± sd, n = 36,*<i>p</i> < 0.05.</p

Effect of 1α,25VitD3 on the expression of phospho-p38 MAPK in the peripheral blood neutrophil of the AECOPD patients.

<p>Neutrophils (1×10⁶) were pretreated with SB203580 (2.5 μM×10^–5) for 0.5 h, followed by incubation with 1α,25VitD3 (6 ×10^-7M) for 24 h. Western blot was used to analyze the phospho-p38 MAPK and p38 MAPK levels. The experiments shown are representative of at least 3 experiments. *<i>p</i> < 0.05.</p